Compositions and methods for treating malaria with cupredoxin and cytochrome

ABSTRACT

The present invention relates to cupredoxin and cytochrome and their use, separately or together, to inhibit the spread of parasitemia in mammalian red blood cells and other tissues infected by the malaria parasite, and in particular the parasitemia of human red blood cells by  P. falciparum . The invention provides isolated peptides that are variants, derivatives or structural equivalents of cupredoxins or cytochrome c, and compositions comprising cupredoxins and/or cytochrome c, or variants, derivatives or structural equivalents thereof, that are useful for treating or preventing malaria infection in mammals. Further, the invention provides methods to treat mammalian patients to prevent or inhibit the growth of malarial infection in mammals. The invention also provides methods to prevent the growth of malaria infection in insect vectors.

RELATED APPLICATIONS

This application is a divisional and claims the benefit under 35 U.S.C.§121, of U.S. patent application Ser. No. 11/436,590, filed May 19,2006, which issued as U.S. Pat. No. 7,338,766 on Mar. 4, 2008, and whichclaims priority to co-filed U.S. Provisional Patent Application Ser. No.60/682,833, entitled “Methods for Treating HIV Infection with Cupredoxinand Cytochrome c”, filed May 20, 2005, U.S. Provisional PatentApplication Ser. No. 60/780,868, filed Mar. 10, 2006, U.S. ProvisionalPatent Application Ser. No. 60/682,813, entitled “Methods for TreatingMalaria with Cupredoxin and Cytochrome,” filed May 20, 2005, and U.S.patent application Ser. No. 11/244,105, filed Oct. 6, 2005, which claimspriority to U.S. Provisional Patent Application Ser. No. 60/616,782,filed Oct. 7, 2004, and U.S. Provisional Patent Application Ser. No.60/680,500, filed May 13, 2005, and is a continuation-in-part of U.S.patent application Ser. No. 10/720,603, filed Nov. 24, 2003, whichclaims priority to U.S. Provisional Patent Application Ser. No.60/414,550, filed Aug. 15, 2003, and which is a continuation-in-part ofU.S. patent application Ser. No. 10/047,710, filed Jan. 15, 2002, whichclaims priority to U.S. Provisional Patent Application Ser. No.60/269,133, filed Feb. 15, 2001. The entire content of these priorapplications is fully incorporated herein by reference.

STATEMENT OF GOVERNMENTAL INTEREST

The subject matter of this application has been supported by researchgrants from the National Institutes of Health (NIH), Bethesda, Md.,U.S., (Grant Numbers AI 16790-21, ES 04050-16, AI 45541, CA09432 andN01-CM97567). The government may have certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to cupredoxin and cytochrome and theiruse, separately or in combination, in inhibiting parasitemia of themalaria parasite, and in particular inhibiting parasitemia of Plasmodiumfalciparum in mammalian red blood cells. The invention also relates tovariants and derivatives of cupredoxin and cytochrome that retain theability to inhibit parasitemia by the malaria parasite. Finally, theinvention provides methods to inhibit the spread of malaria infection ininsect vectors.

BACKGROUND

About one quarter of the world's population is exposed to the risk ofmalaria and more than a million people die of malaria each year. Of thefour species of malarial parasites that infect humans, the two majorspecies are Plasmodium falciparum and P. vivax.

The P. falciparum blood stage merozoites bind to and parasitize theerythrocytes using a variety of surface proteins (Cowman et al., FEBSLett. 476:84-88 (2000); Baum et al., J. Biol. Chem. 281:5197-5208(2006)), a major antigenic member of which is called Merozoite SurfaceProtein 1 (MSP1), a 195 kDa protein. MSP1 is present in all theerythrocyte-invasive species of Plasmodium, anchored to the merozoitesurface by a glycosyl-phosphatidylinositol linkage. During early stagesof the erythrocyte invasion process, soon after release from infectederythrocytes, the merozoite MSP1 protein undergoes proteolytic cleavage,producing a C-terminal cleavage product MSP1-42, which subsequentlyundergoes a second cleavage, producing an 11 kDa peptide MSP1-19, whichremains attached to the parasite surface as it enters the erythrocyte.The formation of the cleavage product MSP1-19 is very important forsuccessful invasion by the parasite since inhibition of its proteolyticformation or its neutralization by monoclonal antibodies prevents entryof the parasite to the erythrocytes (Blackman et al., J. Exptl., Med.180:389-393 (1994)).

The MSP1-19 peptide is one of the most important malaria vaccinecandidates available. MSP1-19-specific antibodies from malaria-resistanthuman sera react with the antigen and include a majorerythrocyte-invasion inhibitory component (Holder & Riley, Parasitol.Today, 12: 173-174 (1996); O'Donnell et al., J. Expt. Med. 193:1403-1412(2001)). Serum from donors in malaria-endemic regions usuallydemonstrates strong antibody reactivity towards Pf MSP1-19. (Nwuba etal., Infect. Immun. 70: 5328-5331 (2002))

The monoclonal antibody (mAb) G17.12 was raised against recombinant PfMSP1-19 and recognizes its epitope on the parasite surface,demonstrating that this region of the antigen is accessible on thenative MSPI polypeptide complex (Pizarro et al., J. Mol. Biol.328:1091-1103 (2003)). Interestingly, erythrocyte invasion experimentsin vitro showed that infection is not inhibited in the presence ofG17.12, even at 200 μg/ml concentration and G17.12 does not inhibit invitro secondary processing of MSP1. Id. The presence of antibodies thatblock the binding of invasion—inhibitory antibodies, therebyfacilitating parasite survival, has also been demonstrated (GuevaraPatino et al., J. Expt. Med. 186: 1689-1699 (1997)), and may beresponsible for the failure of G17.12 mAb to inhibit erythrocyteinvasion by P. falciparum.

Cerebral malaria, a rare but fatal infection restricted to P. falciparuminvasion of brain capillaries because of the sequestration ofparasitized erythrocytes, is often untreatable because most drugs cannotcross the blood-brain barrier to reach the brain capillaries. Adhesionof P. falciparum-infected erythrocytes to brain capillaries is mediatedby the interaction of parasite ligands Pf Emp-1 family of proteinsexpressed on the surface of infected erythrocytes with ICAM-1 and CD36expressed on the surface of capillary endothelium cells in cerebralvessels. (Smith et al., Proc. Natl. Acad. Sci. USA 97:1766-1771 (2000);Franke-Fayard et al., Proc. Natl. Acad. Sci. USA 102, 11468-11473(2005))

Although a few drugs, such as chloroquine that targets the hemedetoxification pathway, are used to treat malaria, there are increasingincidence of parasite resistance to drugs and mosquito vector resistanceto insecticides. Chloroquine antagonizes heme polymerization mediated byparasite-induced HRPs (histidine-rich proteins), as heme monomers arehighly toxic for malaria parasites. The polymerization of heme allowsdetoxification, which is reversed by chloroquine. Another drug,artemisinin, is effective against chloroquine-resistant P. falciparum incerebral malaria. Artemisinin forms adducts with globin-bound heme inhemoglobin, which binds HRPs to prevent heme polymerization. There is anurgent need to find new drugs for this dreaded disease that isparticularly prevalent in Africa and Asia. Present attempts at drugdevelopment are directed towards deciphering the complete parasitegenome sequence, molecular modeling of the malaria parasite proteins anda search for novel drug targets.

SUMMARY OF THE INVENTION

The present invention relates to cupredoxin and cytochrome and theiruse, separately or together, to inhibit the spread of parasitemia inmammalian red blood cells and other tissues infected by the malariaparasite, and in particular the parasitemia of human red blood cells byP. falciparum.

One aspect of the invention is an isolated peptide that is a variant,derivative or structural equivalent of a cupredoxin or cytochrome; andthat can inhibit intracellular replication of a malarial parasite inmalaria-infected human red blood cells.

Another aspect of the invention is an isolated peptide that is avariant, derivative or structural equivalent of a cupredoxin; and thatcan bind a protein selected from the group consisting of PfMSP1-19 andPfMSP1-42.

Another aspect of the invention is an isolated peptide that is avariant, derivative or structural equivalent of a cupredoxin orcytochrome, and that can inhibit parasitemia by malaria inmalaria-infected red blood cells. Specifically, the isolated peptide caninhibit parasitemia by malaria in P. falciparum-infected human red bloodcells. In some embodiments, the cupredoxin is an azurin, pseudoazurin,plastocyanin, rusticyanin, Laz or auracyanin. Specifically, thecupredoxin may be rusticyanin, azurin or Laz. In some embodiments, thecupredoxin is from Pseudomonas aeruginosa, Alcaligenes faecalis,Achromobacter xylosoxidan, Bordetella bronchiseptica, Methylomonas sp.,Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas fluorescens,Pseudomonas chlororaphis, Xylella faslidiosa or Vibrio parahaemolyticus.Specifically, the cupredoxin may be from Thiobacillus ferrooxidans,Pseudomonas aeruginosa, Neisseria gonorrhea or Neisseria meningitidis.

In other embodiments of this aspect, the cytochrome is cytochrome c orcytochrome f. In particular, the cytochrome c may be from human orPseudomonas aeruginosa. The cytochrome f may be from a cyanobacteria.

In other embodiments of this aspect, the isolated peptide is atruncation of a peptide selected from the group consisting of SEQ IDNOS: 1-20 and 22. In some embodiments, SEQ ID NOS: 1-20 or 22 has atleast 90% amino acid sequence identity to the sequence of the isolatedpeptide.

In some embodiments of this aspect, the isolated peptide is a truncationof cupredoxin or cytochrome. In some embodiments, the isolated peptideis more than about 10 residues and not more than about 100 residues. Theisolated peptide may comprise azurin residues 36-89. Alternatively, theisolated peptide may consist of azurin residues 36-89. Alternatively,the isolated peptide may comprise equivalent residues of a cupredoxin asazurin 36-89.

In other embodiments of this aspect, the isolated peptide is fused to aH.8 region of Laz. In another embodiment, the isolated peptide is astructural equivalent of monoclonal antibody G17.12.

Another aspect of the invention is a composition comprising at least onecupredoxin, cytochrome, or isolated peptide that is a variant,derivative or structural equivalent of a cupredoxin or cytochrome thatcan inhibit parasitemia by malaria in malaria-infected red blood cells,in a pharmaceutical composition. Specifically, the pharmaceuticalcomposition may be formulated for intravenous administration. Thecomposition may comprise another anti-malarial drug or an anti-HIV drug.

In some embodiments of the composition, the cupredoxin is fromPseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidan,Bordetella bronchiseptica, Methylomonas sp., Neisseria meningitidis,Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis,Xylella fastidiosa or Vibrio parahaemolyticus. Specifically, thecupredoxin may be from Thiobacillus ferrooxidans, Pseudomonasaeruginosa, Neisseria gonorrhea or Neisseria meningitidis.

In some embodiments of the composition, the cytochrome is cytochrome cor cytochrome f. Specifically, the cytochrome c may be from human orPseudomonas aeruginosa. The cytochrome f may be from a cyanobacteria. Insome embodiments, the cupredoxin or cytochrome c is SEQ ID NOS: 1-20 or22.

Another aspect of the invention is a method to treat a patient sufferingfrom an infection by a malaria parasite by administering to the patientan effective amount of the composition of the invention. In specificembodiments, the peptide inhibits parasitemia by malaria in thepatient's malaria-infected human red blood cells. In some embodiments,the malaria parasite is Plasmodium vivax or Plasmodium falciparum. Insome embodiments, the patient is additionally suffering from HIVinfection. In some embodiments, the composition is administered with asecond composition that may contain an anti-malarial drug and/or ananti-HIV drug. In some embodiments, the composition of the invention isadministered within 0 minutes to 12 hours of the administration ofsecond composition. In some embodiments, the composition of theinvention is administered to the patient orally, by inhalation,intravenously, intramuscularly or subcutaneously; and, specifically, thecomposition may be administered to the patient intravenously.

Another aspect of the invention is a method to treat a patient suspectedof having contact with a malaria parasite, comprising administering tothe patient an effective amount of the composition of the invention.

Another aspect of the invention is a method to prevent malaria inmammals, comprising administering to an insect vector in a population ofinsect vectors harboring a malaria parasite an amount of the compositionof the invention. In some embodiments of this method, the composition isadministered to the insect vector orally.

These and other aspects, advantages, and features of the invention willbecome apparent from the following figures and detailed description ofthe specific embodiments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts surface plasmon resonance binding titrations depictingthe interactions of Azurin, H.8-azurin (H.8-Az), Laz, and GST-azurin(GST-Azu) constructs with MSP1-19 and MSP1-42. (A) Binding curvesdemonstrating the interactions of azurin and its analogues with MSP1-19immobilized on carboxymethyldextran coated gold sensor chips(MSP1-19-CM5). Concentration dependent binding of the azurin proteins toMSP1-19 was determined via injection of various concentrations (0.05-300nM) over the sensor surface and the extent of binding was evaluated as afunction of the equilibrium resonance response value measured inresonance units (RU). While H.8-Az and Laz bound somewhat more stronglythan azurin, no binding was seen with GST or H.8-GST. (B) In vitrobinding titrations for immobilized MSP1-42 with azurin and its analogueswas followed in a similar manner to that for MSP1-19 as shown in (A).Relative binding affinities were determined via fitting the data toReq=Rmax/(1+(Kd/C)) with the curve fits connecting the data points inthe graphs. The MSP1-19 binding Kd values are: 32.2±2.4 nM (azurin),26.2±2.4 nM (Laz), 11.8±0.3 nM (H.8-Az), and those for MSP1-42 bindingare: 54.3±7.6 nM (azurin), 45.6±2.4 nM (Laz) and 14.3±1.7 nM (H.8-Az).(C) Binding titrations for the interactions of GST-Azu fusion proteinsover the MSP1-19-CM5 sensors surface demonstrate the recognition ofGST-Azu 36-128 and GST-Azu 36-89 with MSP1-19. No binding was seen withGST or GST-Azu 88-113.

FIG. 2 depicts inhibition of P. falciparum parasitemia (parasite growthwithin the RBC) by different concentrations, as shown, of Azurin,H.8-azurin (H.8-Az) and Laz. In these experiments, normal red bloodcells were infected with schizonts in absence or in presence of theproteins at different concentrations, incubated overnight and the numberof intracellular parasites was scored by thin blood smear and Giemsastaining.

FIG. 3 depicts surface plasmon resonance binding curves for the bindingof ICAMs (ICAM-1, ICAM-2, ICAM-3 and NCAM, inset) with immobilizedazurin. Due to large nonspecific binding to the bare Au-CM5 chip, CM5was added as an eluent to the running buffer (1 mg/ml CM5 to HBS-EPbuffer). The selective recognition of azurin with ICAM-3, but not withICAM-1 or ICAM-2, is notable and the binding strength was 19.5±5.4 nM.The Kd for NCAM binding with azurin, as shown in the inset, was 20±5.0nM.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is the amino acid sequence of azurin from Pseudomonasaeruginosa.

SEQ ID NO: 2 is the amino acid sequence of cytochrome c₅₅₁ fromPseudomonas aeruginosa.

SEQ ID NO: 3 is the amino acid sequence of Laz from Neisseriameningitidis MC58.

SEQ ID NO: 4 is the amino acid sequence of plastocyanin from Phormidiumlaminosum.

SEQ ID NO: 5 is the amino acid sequence of rusticyanin from Thiobacillusferrooxidans (Acidithiobacillus ferrooxidans).

SEQ ID NO: 6 is the amino acid sequence of pseudoazurin fromAchromobacter cycloclastes.

SEQ ID NO: 7 is the amino acid sequence of azurin from Alcaligenesfaecalis.

SEQ ID NO: 8 is the amino acid sequence of azurin from Achromobacterxylosoxidans ssp. denitrificans I.

SEQ ID NO: 9 is the amino acid sequence of azurin from Bordetellabronchiseptica.

SEQ ID NO: 10 is the amino acid sequence of azurin from Methylomonas sp.J.

SEQ ID NO: 11 is the amino acid sequence of azurin from Neisseriameningitidis Z2491.

SEQ ID NO: 12 is the amino acid sequence of azurin from Pseudomonasfluorescens.

SEQ ID NO: 13 is the amino acid sequence of azurin from Pseudomonaschlororaphis.

SEQ ID NO: 14 is the amino acid sequence of azurin from Xylellafastidiosa 9a5c.

SEQ ID NO: 15 is the amino acid sequence of stellacyanin from Cucumissativus

SEQ ID NO: 16 is the amino acid sequence of auracyanin A fromChloroflexus aurantiacus

SEQ ID NO: 17 is the amino acid sequence of auracyanin B fromChloroflexus aurantiacus

SEQ ID NO: 18 is the amino acid sequence of cucumber basic protein fromCucumis sativus

SEQ ID NO: 19 is the amino acid sequence of cytochrome c from Homosapiens.

SEQ ID NO: 20 is the amino acid sequence of cytochrome f fromcyanobacteria Phormidium laminosum.

SEQ ID NO: 21 is the amino acid sequence of the H.8 region of Laz fromNeisseria gonorrhoeae F62.

SEQ ID NO: 22 is the amino acid sequence of Laz from Neisseriagonorrhoeae F62.

SEQ ID NO: 23 is the forward primer to PCR amplify the Laz-encoding gene(m/z) of Neisseria gonorrhoeae.

SEQ ID NO: 24 is the reverse primer to PCR amplify the Laz-encoding gene(m/z) of Neisseria gonorrhoeae.

SEQ ID NO: 25 is the forward primer to PCR amplify a 3.1 kb fragment ofpUC18-laz.

SEQ ID NO: 26 is the reverse primer to PCR amplify a 3.1 kb fragment ofpuc18-laz.

SEQ ID NO: 27 is the forward primer to PCR amplify a 0.4 kb fragment ofpUC19-paz.

SEQ ID NO: 28 is the reverse primer to PCR amplify a 0.4 kb fragment ofpUC19-paz.

SEQ ID NO: 29 is the forward primer for pGST-azu 36-128.

SEQ ID NO: 30 is the reverse primer for pGST-azu 36-128.

SEQ ID NO: 31 is the forward primer for pGST-azu 3689.

SEQ ID NO: 32 is the reverse primer for pGST-azu 36-89.

SEQ ID NO: 33 is the forward primer for pGST-azu 88-113.

SEQ ID NO: 34 is the reverse primer for pGST-azu 88-113.

SEQ ID NO: 35 is an oligonucleotide for site directed mutagenesis forthe preparation of pGST-azu 88-113.

SEQ ID NO: 36 is an oligonucleotide for site directed mutagenesis forthe preparation of pGST-azu 88-113.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the term “cell” includes both the singular or the pluralof the term, unless specifically described as a “single cell.”

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid. The terms also apply to naturally occurring aminoacid polymers. The terms “polypeptide,” “peptide,” and “protein” arealso inclusive of modifications including, but not limited to,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation. It will beappreciated that polypeptides are not always entirely linear. Forinstance, polypeptides may be branched as a result of ubiquitination andthey may be circular (with or without branching), generally as a resultof post-translation events, including natural processing event andevents brought about by human manipulation which do not occur naturally.Circular, branched and branched circular polypeptides may be synthesizedby non-translation natural process and by entirely synthetic methods aswell.

As used herein, the term “pathological condition” includes anatomic andphysiological deviations from the normal that constitute an impairmentof the normal state of the living animal or one of its parts, thatinterrupts or modifies the performance of the bodily functions, and is aresponse to various factors (as malnutrition, industrial hazards, orclimate), to specific infective agents (as worms, parasitic protozoa,bacteria, or viruses), to inherent defects of the organism (as geneticanomalies), or to combinations of these factors.

As used herein, the term “condition” includes anatomic and physiologicaldeviations from the normal that constitute an impairment of the normalstate of the living animal or one of its parts, that interrupts ormodifies the performance of the bodily functions.

As used herein, the term “suffering from” includes presently exhibitingthe symptoms of a pathological condition, having a pathologicalcondition even without observable symptoms, in recovery from apathological condition, or recovered from a pathological condition.

As used herein, the term “parasitemia” includes a condition in whichparasites are present in the blood and other tissues, and in particularto indicate the presence of parasites with or without clinical symptoms.

As used herein, the term “inhibition of parasitemia” refers to adecrease or a lessening of the rate of increase of the presence of theparasite in the blood of a mammal. Inhibition is any decrease orlessening of the rate of increase that is statistically significant ascompared to control treatments.

As used herein, the term “treatment” includes preventing, lowering,stopping, or reversing the progression or severity of the condition orsymptoms associated with a condition being treated. As such, the term“treatment” includes medical, therapeutic, and/or prophylacticadministration, as appropriate.

As used herein, “anti-malarial activity” includes any activity thatdecreases the infectivity, the reproduction, or inhibits the progress ofthe lifecycle of a malaria parasite. “Anti-malarial activity” includesinhibition of the growth of malaria infection by all of the means ofobserved with current anti-malarial drugs.

As used herein, the term “anti-malarial drug” refers to drugs withanti-malarial activity that may be used to decrease the infectivity, thereproduction, or inhibit the progress of the lifecycle of a malariaparasite.

As used herein, the term “anti-HIV drug” refers to drugs with anti-HIVactivity HIV by which HIV infection in mammals is decreased, orprevented from increasing in the human body, by any means including, butare not limited to, inhibition of replication of the HIV genome,inhibition of synthesis and/or assembly of the HIV coat proteins, andinhibition of HIV entry into uninfected cells.

The term “substantially pure”, when used to modify the term apolypeptide or other compound, as used herein, refers to a polypeptideor compound, for example, a polypeptide isolated from the growth medium,in a form substantially free of, or unadulterated by, active inhibitoryagents. The term “substantially pure” refers to a compound in an amountof at least about 75%, by dry weight, of isolated fraction, or “75%substantially pure.” More specifically, the term “substantially pure”refers to a compound of at least about 85%, by dry weight, activecompound, or “85% substantially pure.” Most specifically, the term“substantially pure” refers to a compound of at least about 95%, by dryweight, active compound, or “95% substantially pure.” The substantiallypure cupredoxin or cytochrome c₅₅₁ or a variant or derivative thereofcan be used in combination with one or more other substantially purecompounds, or another isolated cupredoxin or cytochrome.

The phrases “isolated,” “purified” or “biologically pure” refer tomaterial which is substantially or essentially free from componentswhich normally accompany the material as it is found in its nativestate. Thus, isolated peptides in accordance with the inventionpreferably do not contain materials normally associated with thepeptides in their in situ environment. An “isolated” region refers to aregion that does not include the whole sequence of the polypeptide fromwhich the region was derived. An “isolated” nucleic acid, protein, orrespective fragment thereof has been substantially removed from its invivo environment so that it may be manipulated by the skilled artisan,such as but not limited to nucleotide sequencing, restriction digestion,site-directed mutagenesis, and subcloning into expression vectors for anucleic acid fragment as well as obtaining the protein or proteinfragment in substantially pure quantities.

The term “variant” as used herein with respect to a peptide, refers toamino acid sequence variants which may have amino acids replaced,deleted, or inserted as compared to the wild-type polypeptide. Variantsmay be truncations of the wild-type peptide. Thus, a variant peptide maybe made by manipulation of genes encoding the polypeptide. A variant maybe made by altering the basic composition or characteristics of thepolypeptide, but not at least some of its fundamental activities. Forexample, a “variant” of azurin can be a mutated azurin that retains itsability to inhibit parasitemia in malaria-infected human red bloodcells. In some cases, a variant peptide is synthesized with non-naturalamino acids, such as ε-(3,5-dinitrobenzoyl)-Lys residues. (Ghadiri &Femholz, J. Am. Chem. Soc., 112:9633-9635 (1990)) In some embodiments,the variant has not more than 20 amino acids replaced, deleted orinserted compared to wild-type peptide. In some embodiments, the varianthas not more than 15, 14, 13, 12 or 11 amino acids replaced, deleted orinserted compared to wild-type peptide. In some embodiments, the varianthas not more than 10, 9, 8 or 7 amino acids replaced, deleted orinserted compared to wild-type peptide. In some embodiments, the varianthas not more than 6 amino acids replaced, deleted or inserted comparedto wild-type peptide. In some embodiments, the variant has not more than5 or 4 amino acids replaced, deleted or inserted compared to wild-typepeptide. In some embodiments, the variant has not more than 3, 2 or 1amino acids replaced, deleted or inserted compared to wild-type peptide.

The term “amino acid,” as used herein, means an amino acid moiety thatcomprises any naturally-occurring or non-naturally occurring orsynthetic amino acid residue, i.e., any moiety comprising at least onecarboxyl and at least one amino residue directly linked by one, twothree or more carbon atoms, typically one (a) carbon atom.

The term “derivative” as used herein with respect to a peptide refers toa peptide that is derived from the subject peptide. A derivationincludes chemical modifications of the peptide such that the peptidestill retains some of its fundamental activities. For example, a“derivative” of azurin can be a chemically modified azurin that retainsits ability to inhibit parasitemia in malaria-infected red blood cells.Chemical modifications of interest include, but are not limited to,amidation, acetylation, sulfation, polyethylene glycol (PEG)modification, phosphorylation or glycosylation of the peptide. Inaddition, a derivative peptide may be a fusion of a polypeptide orfragment thereof to a chemical compound, such as but not limited to,another peptide, drug molecule or other therapeutic or pharmaceuticalagent or a detectable probe.

The term “percent (%) amino acid sequence identity” is defined as thepercentage of amino acid residues in a polypeptide that are identicalwith amino acid residues in a candidate sequence when the two sequencesare aligned. To determine % amino acid identity, sequences are alignedand if necessary, gaps are introduced to achieve the maximum % sequenceidentity; conservative substitutions are not considered as part of thesequence identity. Amino acid sequence alignment procedures to determinepercent identity are well known to those of skill in the art. Oftenpublicly available computer software such as BLAST, BLAST2, ALIGN2 orMegalign (DNASTAR) software is used to align peptide sequences. In aspecific embodiment, Blastp (available from the National Center forBiotechnology Information, Bethesda Md.) is used using the defaultparameters of long complexity filter, expect 10, word size 3, existence11 and extension 1.

When amino acid sequences are aligned, the % amino acid sequenceidentity of a given amino acid sequence A to, with, or against a givenamino acid sequence B (which can alternatively be phrased as a givenamino acid sequence A that has or comprises a certain % amino acidsequence identity to, with, or against a given amino acid sequence B)can be calculated as:% amino acid sequence identity=X/Y*100

where

-   -   X is the number of amino acid residues scored as identical        matches by the sequence alignment program's or algorithm's        alignment of A and B and    -   Y is the total number of amino acid residues in B.

If the length of amino acid sequence A is not equal to the length ofamino acid sequence B, the % amino acid sequence identity of A to B willnot equal the % amino acid sequence identity of B to A. When comparinglonger sequences to shorter sequences, the shorter sequence will be the“B” sequence, unless stated otherwise. For example, when comparingtruncated peptides to the corresponding wild-type polypeptide, thetruncated peptide will be the “B” sequence.

A “therapeutically effective amount” is an amount effective to preventor slow the development of, or to partially or totally alleviate theexisting symptoms in a particular condition, pathological or otherwise,for which the subject being treated. Determination of a therapeuticallyeffective amount is well within the capability of those skilled in theart.

General

The present invention provides compositions and methods that usecupredoxin and/or cytochrome to inhibit parasitemia of malaria-infectedmammalian red blood cells and bodily tissues, such as brain tissue andbone tissue.

Previously it was known that several bacterial redox proteins belongingto a family of the blue copper-containing proteins called cupredoxins,or the iron (haem)-containing proteins called cytochromes, entermammalian cells, including cancer cells, and either induce apoptoticcell death or cause growth inhibition through G1 arrest of the cellcycle. (Yamada et al., Cell Cycle 3:752-755 (2004); Yamada et al., CellCycle 3:1182-1187 (2004)) Single bacterial proteins such as thecupredoxin azurin or the cytochrome c₅₅₁, both elaborated by Pseudomonasaeruginosa, can demonstrate either activity based on theirhydrophobicity. Thus wild type (wt) azurin induces apoptosis in themurine J774 cells (Yamada et al., Infection and Immunity 70:7054-7062(2002)) while a mutant M44KM64E azurin causes cell cycle inhibition atthe G1 phase in J774 cells. (Yamada et al., PNAS 101:4770-4775 (2004))In contrast, wt cytochrome c₅₅₁ causes cell cycle inhibition at the G1phase in J774 cells while a mutant V23D159E cytochrome c₅₅₁ inducesapoptosis. (Hiraoka et al., PNAS 101:6427-6432 (2004))

In accordance with the present invention, it is surprisingly now knownthat cupredoxins and cytochromes will inhibit in vitro parasitemia inhuman red blood cells by the malaria parasite Plasmodium falciparum. Inparticular, the cupredoxins azurin and Laz inhibit parasitemia in P.falciparum by about 50% and about 75% respectively. See, Example 6.Further, rusticyanin and cytochromes c and f inhibited parasitemia by20-30%. See, Example 1. Further, it is now known that azurin has adiscernable structural homology to the Fab fragment of G17.12 mousemonoclonal antibody when complexed to the Pf MSP1-9 fragment of the MSP1surface protein of P. falciparum. See, Example 2. While not limiting themode of inhibition to any one means, it is thought that azurin mayinhibit parasitemia of P. falciparum by interaction with the MSP1protein on the parasite's surface.

Surprisingly, it is now known that azurin and Laz bind both thePfMSP1-19 and PfMSP1-42 P. falciparum surface proteins in vitro.Further, it is now known that azurin amino acid residues 36-89 arerequired for binding to PfMSP1-19 and PfMSP1-42. Further, it is nowknown that the H.8 domain of Laz from N. gonorrhea increases both thebinding of a fused azurin to PfMSP1-19 as well as inhibition ofparasitemia by P. falciparum. See, Examples 5 and 6.

Because of the high structural homology between the cupredoxins, it iscontemplated that other cupredoxins will have the same anti-malarialactivity as the azurin, rusticyanin, or Laz. In some embodiments, thecupredoxin is, but is not limited to, azurin, pseudoazurin,plastocyanin, auracyanin, Laz or rusticyanin. In specific embodiments,the cupredoxin is Laz, azurin or rusticyanin. In other embodiments, thecupredoxin is from a pathogenic bacteria. In a more specific embodimentthe cupredoxin is azurin. In particularly specific embodiments, theazurin is derived from Pseudomonas aeruginosa, Alcaligenes faecalis,Achromobacter xylosoxidans ssp. denitrificans I., Bordetellabronchiseptica, Methylomonas sp., Neisseria meningitidis Z2491,Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylella fastidiosa9a5 or Vibrio parahaemolyticus. In a most specific embodiment, theazurin is from P. aeruginosa. In other specific embodiments, thecupredoxin comprises an amino acid sequence that is SEQ ID NO: 1, 2-18or 21.

In accordance with the present invention, it has been learned that P.aeruginosa cytochrome c₅₅₁, human cytochrome c and Phormidium laminosumcytochrome f will inhibit parasitemia in malaria-infected human redblood cells. In a specific embodiment, the cytochrome is cytochrome c₅₅₁from P. aeruginosa, human cytochrome c or cytochrome f. In otherspecific embodiments, the cytochrome comprises an amino acid sequencethat is SEQ ID NO: 2, 19 or 20.

Because of the structural homology between the cytochrome c's, it iscontemplated that other cytochromes will have the same anti-malarialactivity as P. aeruginosa cytochrome c₅₅₁ and human cytochrome c. Insome embodiments, the cytochrome is from a pathogenic bacterium. Inanother specific embodiment, the cytochrome inhibits parasitism inmalaria-infected red blood cells, and more specifically, human red bloodcells. In another specific embodiment, the cytochrome inhibits cellcycle progression in a mammalian cancer cell, and more specifically in aJ774 cell.

Compositions of the Invention

The invention provides for peptides that are variants, derivatives orstructural equivalents of cupredoxin or cytochrome. In some embodiments,the peptide is substantially pure. In other embodiments, the peptide isisolated. In some embodiments, the peptide is less that a full lengthcupredoxin or cytochrome, and retains some of the functionalcharacteristics of the cupredoxin or cytochrome. In some embodiments,the peptide retains the ability to inhibit parasitemia inmalaria-infected red blood cells, and more specifically the ability toinhibit P. falciparum infection in human red blood cells. In specificembodiments, the cytochrome is P. aeruginosa cytochrome c₅₅₁, humancytochrome c, or cyanobacterial cytochrome f, and specifically SEQ IDNOS: 2, 19, and 20. In another specific embodiment, the peptide does notraise an immune response in a mammal, and more specifically a human.

The invention also provides compositions comprising at least one peptidethat is a cupredoxin, cytochrome, or variant, derivative or structuralequivalent of a cupredoxin or cytochrome. The invention also providescompositions comprising at least one peptide that is a cupredoxin orvariant, derivative or structural equivalent of a cupredoxin. Theinvention also provides compositions comprising at least one peptidethat is a cytochrome, or variant, derivative or structural equivalent ofa cytochrome. In other embodiments, the composition consists essentiallyof the peptide. The invention also provides compositions comprising atleast one peptide that is a cupredoxin, cytochrome, or variant,derivative or structural equivalent of a cupredoxin or cytochrome in apharmaceutical composition.

Because of the high structural homology between the cupredoxins, it iscontemplated that other cupredoxins will have the sane anti-malarialactivity as Pseudomonas aeruginosa azurin with regards to inhibition ofparasitemia in malaria-infected red blood cells. In some embodiments,the cupredoxin is, but is not limited to, azurin, pseudoazurin,plastocyanin, rusticyanin, Laz or auracyanin. In particularly specificembodiments, the cupredoxin is derived from Pseudomonas aeruginosa,Alcaligenes faecalis, Achromobacter xylosoxidans ssp. denitrificans I,Bordetella bronchiseptica, Methylomonas sp., Neisseria meningitidisZ2491, Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonaschlororaphis, Xylella fastidiosa 9a5 or Vibrio parahaemolyticus. In avery specific embodiment, the cupredoxin is azurin from Pseudomonasaeruginosa. In other specific embodiments, the cupredoxin comprises anamino acid sequence that is SEQ ID NO: 1, 3-18, or 22. In other specificembodiments, the cupredoxin is the Laz protein from Neisseriameningitidis or Neisseria gonorrhea.

The invention provides for amino acid sequence variants of a cupredoxinor cytochrome which have amino acids replaced, deleted, or inserted ascompared to the wild-type polypeptide. Variants of the invention may betruncations of the wild-type polypeptide. In some embodiments, thecomposition comprises a peptide that consists of a region of acupredoxin or cytochrome that is less that the full length wild-typepolypeptide. In some embodiments, the composition comprises a peptidethat consists of more than about 10 residues, more than about 15residues or more than about 20 residues of a truncated cupredoxin orcytochrome. In some embodiments, the composition comprises a peptidethat consists of not more than about 100 residues, not more than about50 residues, not more than about 40 residues or not more than about 30residues of a truncated cupredoxin or cytochrome. In some embodiments,composition comprises a peptide to which a cupredoxin or cytochrome, andmore specifically to SEQ ID NOS: 1-20 or 22 has at least about 90% aminoacid sequence identity, at least about 95% amino acid sequence identityor at least about 99% amino acid sequence identity.

In specific embodiments, the variant of cupredoxin comprises P.aeruginosa azurin residues 36-89. In other embodiments, the variant ofcupredoxin consists of P. aeruginosa azurin residues 36-89. In otherspecific embodiments, the variant consists of the equivalent residues ofa cupredoxin other that azurin.

It is contemplated that other cupredoxin variants can be designed thathave a similar activity to azurin residues 36-89. To do this, thesubject cupredoxin amino acid sequence will be aligned to thePseudomonas aeruginosa azurin sequence using BLAST, BLAST2, ALIGN2 orMegalign (DNASTAR), the relevant residues located on the P. aeruginosaazurin amino acid sequence, and the equivalent residues found on thesubject cupredoxin sequence, and the equivalent residues of thecupredoxin thus identified.

The variants also include peptides made with synthetic amino acids notnaturally occurring. For example, non-naturally occurring amino acidsmay be integrated into the variant peptide to extend or optimize thehalf-life of the composition in the bloodstream. Such variants include,but are not limited to, D,L-peptides (diastereomer), (Futaki et al., J.Biol. Chem. 276(8):5836-40 (2001); Papo et al., Cancer Res.64(16):5779-86 (2004); Miller et al, Biochem. Pharmacol. 36(1):169-76,(1987)); peptides containing unusual amino acids (Lee et al., 1. Pept.Res. 63(2):69-84 (2004)), and incorporation of olefin-containingnon-natural amino acid followed by hydrocarbon stapling (Schafmeister etal., J. Am. Chem. Soc. 122:5891-5892 (2000); Walenski et al., Science305:1466-1470 (2004)) and peptides comprising ε-(3,5-dinitrobenzoyl)-Lysresidues.

In other embodiments, the peptide of the invention is a derivative of acupredoxin or cytochrome. The derivatives of cupredoxin or cytochromeare chemical modifications of the peptide such that the peptide stillretains some of its fundamental activities. For example, a “derivative”of azurin can be a chemically modified azurin that retains its abilityto inhibit the malaria parasitemia in mammalian cells. Chemicalmodifications of interest include, but are not limited to, amidation,acetylation, sulfation, polyethylene glycol (PEG) modification,phosphorylation and glycosylation of the peptide. In addition, aderivative peptide may be a fusion of a cupredoxin or cytochrome, orvariant derivative or structural equivalent thereof to a chemicalcompound, such as but not limited to, another peptide, drug molecule orother therapeutic or pharmaceutical agent or a detectable probe.Derivatives of interest include chemical modifications by which thehalf-life in the bloodstream of the peptides and compositions of theinvention can be extended or optimized, such as by several methods wellknown to those in the art, including but not limited to, circularizedpeptides (Monk et al., BioDrugs 19(4):261-78, (2005); DeFreest et al.,J. Pept. Res. 63(5):409-19 (2004)), N- and C-terminal modifications(Labrie et al., Clin. Invest. Med. 13(5):275-8, (1990)), andincorporation of olefin-containing non-natural amino acid followed byhydrocarbon stapling (Schafmeister et al., J. Am. Chem. Soc.122:5891-5892 (2000); Walenski et al., Science 305:1466-1470 (2004)).

In one embodiment, the cupredoxin or cytochrome, or variant, derivativeor structural equivalent thereof, is fused to a H.8 region of Laz fromNeisseria meningitidis or Neisseria gonorrhea. One example of such apeptide is the H.8-Paz fusion protein described in Example 4. In aspecific embodiment, the H.8 is fused to the C-terminus of thecupredoxin or cytochrome, or variant, derivative or structuralequivalent thereof. In another specific embodiment, the H.8 region isSEQ ID NO: 21, or a variant, derivative or structural equivalentthereof.

It is contemplated that the peptide of the composition of invention maybe more than one of a variant, derivative and structural equivalent of acupredoxin or cytochrome. For example, the peptide may be a truncationof azurin that has been PEGylated, thus making it both a variant and aderivative. In one embodiment, the peptides of the invention aresynthesized with α,α-disubstituted non-natural amino acids containingolefin-bearing tethers, followed by an all-hydrocarbon “staple” byruthenium catalyzed olefin metathesis. (Scharmeister et al., J. Am.Chem. Soc. 122:5891-5892 (2000); Walensky et al., Science 305:1466-1470(2004)) Additionally, peptides that are structural equivalents of azurinmay be fused to other peptides, thus making a peptide that is both astructural equivalent and a derivative. These examples are merely toillustrate and not to limit the invention. Variants, derivatives orstructural equivalents of cupredoxin or cytochrome may or may not bindcopper.

In another embodiment, the peptide may be a structural equivalent of acupredoxin or cytochrome. Examples of studies that determine significantstructural homology between cupredoxins and cytochromes and otherproteins include Toth et al. (Developmental Cell 1:82-92 (2001)).Specifically, significant structural homology between a cupredoxin orcytochrome and its structural equivalents are determined by using theVAST algorithm (Gibrat et al., Curr Opin Struct Biol 6:377-385 (1996);Madej et al., Proteins 23:356-3690 (1995)). In specific embodiments, theVAST p value from a structural comparison of a cupredoxin or cytochrometo the structural equivalent is less than about 10⁻³, less than about10⁻⁵, or less than about 10⁻⁷. In other embodiments, significantstructural homology between a cupredoxin or cytochrome and itsstructural equivalents are determined by using the DALI algorithm (Holm& Sander, J. Mol. Biol. 233:123-138 (1993)). In specific embodiments,the DALI Z score for a pairwise structural comparison is at least about3.5, at least about 7.0, or at least about 10.0.

In another embodiment, the variant or derivative of cupredoxin has asignificant structural homology to the Fab fragment of G17.12 mousemonoclonal antibody. An example of how this structural similarity can bedetermined can be found in Example 3. Specifically, significantstructural homology between a cupredoxin and the Fab fragment of G17.12mouse monoclonal antibody can be determined by using the VAST algorithm(Gibrat et al., id.; Madej et al., id.). In specific embodiments, theVAST p-value from a structural comparison of a cupredoxin to the Fabfragment of G17.12 mouse monoclonal antibody can be less than about10⁻⁴, less than about 10⁻⁵, less than about 10⁻⁶, or less than about10⁻⁷. In other specific embodiments, the VAST score from a structuralcomparison of a cupredoxin to the Fab fragment of G17.12 mousemonoclonal antibody can be greater than about 9, greater than about 10,greater than about 11 or greater than about 12.

In some embodiments, the variant, derivative or structural equivalentthereof has some of the functional characteristics of the P. aeruginosaazurin, P. aeruginosa cytochrome c₅₅₁, human cytochrome c orcyanobacterial cytochrome f. In a specific embodiment, the peptide ofthe invention inhibits parasitemia by malaria in malaria-infected redblood cells, and more specifically parasitemia by P. falciparum in P.falciparum-infected human red blood cells. The invention also providesfor the variants, derivatives and structural equivalents of cupredoxinand cytochrome c₅₅₁ that retain the ability to inhibit parasitemia inmalaria-infected red blood cells, and more specifically parasitemia byP. falciparum in P. falciparum-infected human red blood cells. Theinhibition of parasitemia by P. falciparum in P. falciparum-infectedhuman red blood cells may be determined by the method described inExample 6.

Because it is now known that cupredoxins and cytochrome can inhibitparasitemia in malaria-infected red blood cells, it is now possible todesign variants, derivatives and structural equivalents of cupredoxinsand cytochrome that retain this anti-malarial activity. Such variantsand derivatives can be made by, for example, creating a “library” ofvarious variants and derivatives of cupredoxins and cytochromes, andthen testing each for anti-malarial activity using one of many methodsknown in the art, such the exemplary method in Example 6. It iscontemplated that the resulting variants, derivatives and structuralequivalents of cupredoxins and cytochromes with anti-malarial activitycan be used in the methods of the invention, in place of or in additionto the cupredoxins and cytochromes mentioned herein. This method ofselecting variants and derivatives may be adapted for any of theactivities of P. aeruginosa azurin, P. aeruginosa cytochrome c₅₅₁, humancytochrome c or cyanobacterial cytochrome f disclosed herein.

In other embodiments, the peptide of the invention inhibitsintracellular replication of the malaria parasite in human red bloodcells. Methods to determine the intracellular replication of the malariaparasite are well known in the art, and one such method is described inExample 2.

In some embodiments, the peptide of the invention binds to the PfMSP1-19and/or PfMSP1-42 P. falciparum surface proteins with a relative bindingaffinity that is statistically greater a non-binding control protein. Apeptide can be tested for this activity by using surface plasmonresonance analysis as described in Example 5. Other methods to determinewhether one protein binds to another are well known in the art and maybe used as well.

In another embodiment, the peptide of the invention binds to ICAM-3 orNCAM with a relative binding affinity that is statistically greater anon-binding control protein. A peptide can be tested for this activityby using surface plasmon resonance analysis as described in Examples 7and 5. Other methods to determine whether one protein binds to anotherare well known in the art and may be used as well.

In some specific embodiments, the peptides of the invention induceapoptosis in a mammalian cancer cell, more specifically a J774 cell. Theability of a peptide to induce apoptosis may be observed by mitosensorApoAlert™ confocal microscopy using a MITOSENSOR™ APOLERT™ MitochondrialMembrane Sensor kit (Clontech Laboratories, Inc., Palo Alto, Calif.,U.S.A.), by measuring caspase-8, caspase-9 and caspase-3 activity usingthe method described in Zou et al. (J. Biol. Chem. 274: 11549-11556(1999)), and by detecting apoptosis-induced nuclear DNA fragmentationusing, for example, the APOLERT™ DNA fragmentation kit (ClontechLaboratories, Inc., Palo Alto, Calif., U.S.A.).

In another specific embodiment, the peptide of the invention inducescellular growth arrest in a mammalian cancer cell, more specifically aJ774 cell. Cellular growth arrest can be determined by measuring theextent of inhibition of cell cycle progression, such as by the methodfound in Yamada et al. (PNAS 101:4770-4775 (2004)). In another specificembodiment, the peptide of the invention inhibits cell cycle progressionin a mammalian cancer cell, more specifically a J774 cell.

Cupredoxins

These small blue copper proteins (cupredoxins) are electron transferproteins (10-20 kDa) that participate in bacterial electron transferchains or are of unknown function. The copper ion is solely bound by theprotein matrix. A special distorted trigonal planar arrangement to twohistidine and one cysteine ligands around the copper gives rise to verypeculiar electronic properties of the metal site and an intense bluecolor. A number of cupredoxins have been crystallographicallycharacterized at medium to high resolution.

The cupredoxins in general have a low sequence homology but highstructural homology. (Gough & Clothia, Structure 12:917-925 (2004); DeRienzo et al., Protein Science 9:1439-1454 (2000).) For example, theamino acid sequence of azurin is 31% identical to that of auracyanin B,16.3% to that of rusticyanin, 20.3% to that of plastocyanin, and 17.3%to that of pseudoazurin. See Table 1. However, the structural similarityof these proteins is more pronounced. The VAST p value for thecomparison of the structure of azurin to auracyanin B is 10^(−7.4),azurin to rusticyanin is 10⁻⁵, azurin to plastocyanin is 10^(−5.6), andazurin to psuedoazurin is 10^(−4.1).

All of the cupredoxins possess an eight-stranded Greek key beta-barrelor beta-sandwich fold and have a highly conserved site architecture. (DeRienzo et al., Protein Science 9:1439-1454 (2000).) A prominenthydrophobic patch, due to the presence of many long chain aliphaticresidues such as methionines and leucines, is present around the coppersite in azurins, amicyanins, cyanobacterial plastocyanins, cucumberbasic protein and to a lesser extent, pseudoazurin and eukaryoticplastocyanins. Id Hydrophobic patches are also found to a lesser extentin stellacyanin and rusticyanin copper sites, but have differentfeatures. Id.

TABLE 1 Sequence and structure alignment of azurin (1JZG) from P.aeruginosa to other proteins using VAST algorithm. Align- ment % aa PDBlength¹ identity P-value² Score³ RMSD⁴ Description 1AOZ A 2 82 18.310e−7 12.2 1.9 Ascorbate oxidase 1QHQ_A 113 31 10e−7.4 12.1 1.9AuracyaninB 1V54 B 1 79 20.3 10e−6.0 11.2 2.1 Cytocrome c oxidase 1GY2 A92 16.3 10e−5.0 11.1 1.8 Rusticyanin 3MSP A 74 8.1 10e−6.7 10.9 2.5Motile Major Sperm Protein⁵ 1IUZ 74 20.3 10e−5.6 10.3 2.3 Plastocyanin1KGY E 90 5.6 10e−4.6 10.1 3.4 Ephrinb2 1PMY 75 17.3 10e−4.1 9.8 2.3Pseudoazurin ¹Aligned Length: The number of equivalent pairs of C-alphaatoms superimposed between the two structures, i.e. how many residueshave been used to calculate the 3D superposition. ²P-VAL: The VAST pvalue is a measure of the significance of the comparison, expressed as aprobability. For example, if the p value is 0.001, then the odds are1000 to 1 against seeing a match of this quality by pure chance. The pvalue from VAST is adjusted for the effects of multiple comparisonsusing the assumption that there are 500 independent and unrelated typesof domains in the MMDB database. The p value shown thus corresponds tothe p value for the pairwise comparison of each domain pair, divided by500. ³Score: The VAST structure-similarity score. This number is relatedto the number of secondary structure elements superimposed and thequality of that superposition. Higher VAST scores correlate with highersimilarity. ⁴RMSD: The root mean square superposition residual inAngstroms. This number is calculated after optimal superposition of twostructures, as the square root of the mean square distances betweenequivalent C-alpha atoms. Note that the RMSD value scales with theextent of the structural alignments and that this size must be takeninto consideration when using RMSD as a descriptor of overall structuralsimilarity. ⁵ C. elegans major sperm protein proved to be an ephrinantagonist in oocyte maturation (Kuwabara, 2003 “The multifaceted C.elegans major sperm protein: an ephrin signalling antagonist in oocytematuration” Genes and Development, 17: 155-161.

Azurin

The azurins are copper containing proteins of 128 amino acid residueswhich belong to the family of cupredoxins involved in electron transferin plants and certain bacteria. The azurins include those from P.aeruginosa (PA) (SEQ ID NO: 1), A. xylosoxidans, and A. denitrificans(SEQ ID NO: 8). (Murphy et al., J. Mol. Biol. 315:859-871 (2002)) Theamino acid sequence identity between the azurins varies between 60-90%,these proteins showed a strong structural homology. All azurins have acharacteristic β-sandwich with Greek key motif and the single copperatom is always placed at the same region of the protein. In addition,azurins possess an essentially neutral hydrophobic patch surrounding thecopper site. Id.

Plastocyanins

The plastocyanins are soluble proteins of cyanobacteria, algae andplants that contain one molecule of copper per molecule and are blue intheir oxidized form. They occur in the chloroplast, where they functionas electron carriers. Since the determination of the structure of poplarplastocyanin in 1978, the structure of algal (Scenedesmus, Enteromorpha,Chlamydomonas) and plant (French bean) plastocyanins has been determinedeither by crystallographic or NMR methods, and the poplar structure hasbeen refined to 1.33 Å resolution. SEQ ID NO: 4 shows the amino acidsequence of plastocyanin from Phormidium laminosum, a thermophiliccyanobacterium.

Despite the sequence divergence among plastocyanins of algae andvascular plants (e.g., 62% sequence identity between the Chlamydomonasand poplar proteins), the three-dimensional structures are conserved(e.g., 0.76 Å nms deviation in the C alpha positions between theChlamydomonas and Poplar proteins). Structural features include adistorted tetrahedral copper binding site at one end of aneight-stranded antiparallel beta-barrel, a pronounced negative patch,and a flat hydrophobic surface. The copper site is optimized for itselectron transfer function, and the negative and hydrophobic patches areproposed to be involved in recognition of physiological reactionpartners. Chemical modification, cross-linking, and site-directedmutagenesis experiments have confirmed the importance of the negativeand hydrophobic patches in binding interactions with cytochrome f, andvalidated the model of two functionally significant electron transferpaths involving plastocyanin. One putative electron transfer path isrelatively short (approximately 4 Å) and involves the solvent-exposedcopper ligand His-87 in the hydrophobic patch, while the other is morelengthy (approximately 12-15 Å) and involves the nearly conservedresidue Tyr-83 in the negative patch, Redinbo et al., J. Bioenerg.Biomembr. 26:49-66 (1994).

Rusticyanins

Rusticyanins are blue-copper containing single-chain polypeptidesobtained from a Thiobacillus (now called Acidithiobacillus). The X-raycrystal structure of the oxidized form of the extremely stable andhighly oxidizing cupredoxin rusticyanin from Thiobacillus ferrooxidans(SEQ ID NO: 5) has been determined by multiwavelength anomalousdiffraction and refined to 10.9 Å resolution. The rusticyanins arecomposed of a core beta-sandwich fold composed of a six- and aseven-stranded b-sheet. Like other cupredoxins, the copper ion iscoordinated by a cluster of four conserved residues (His 85, Cys138,His143, Met148) arranged in a distorted tetrahedron. Walter, R. L. etal., J. Mol. Biol., vol. 263, pp-730-51 (1996).

Pseudoazurins

The pseudoazurins are a family of blue-copper containing single-chainpolypeptide. The amino acid sequence of pseudoazurin obtained fromAchromobacter cycloclastes is shown in SEQ ID NO: 6. The X-ray structureanalysis of pseudoazurin shows that it has a similar structure to theazurins although there is low sequence homology between these proteins.Two main differences exist between the overall structure of thepseudoazurins and azurins. There is a carboxy terminus extension in thepseudoazurins, relative to the azurins, consisting of two alpha-helices.In the mid-peptide region azurins contain an extended loop, shortened inthe pseudoazurins, which forms a flap containing a short t-helix. Theonly major differences at the copper atom site are the conformation ofthe MET side-chain and the Met-S copper bond length, which issignificantly shorter in pseudoazurin than in azurin.

Phytocyanins

The proteins identifiable as phytocyanins include, but are not limitedto, cucumber basic protein, stellacyanin, mavicyanin, umecyanin, acucumber peeling cupredoxin, a putative blue copper protein in pea pods,and a blue copper protein from Arabidopsis thaliana. In all exceptcucumber basic protein and the pea-pod protein, the axial methionineligand normally found at blue copper sites is replaced by glutamine.

Auracyanin

Three small blue copper proteins designated auracyanin A, auracyaninB-1, and auracyanin B-2 have been isolated from the thermophilic greengliding photosynthetic bacterium Chloroflexus aurantiacus. The two Bforms are glycoproteins and have almost identical properties to eachother, but are distinct from the A form. The sodium dodecylsulfate-polyacrylamide gel electrophoresis demonstrates apparent monomermolecular masses as 14 (A), 18 (B-2), and 22 (B-1) kDa.

The amino acid sequence of auracyanin A has been determined and showedauracyanin A to be a polypeptide of 139 residues. (Van Dreissche et al.,Protein Science 8:947-957 (1999).) His58, Cys123, His128, and Met132 arespaced in a way to be expected if they are the evolutionary conservedmetal ligands as in the known small copper proteins plastocyanin andazurin. Secondary structure prediction also indicates that auracyaninhas a general beta-barrel structure similar to that of azurin fromPseudomonas aeruginosa and plastocyanin from poplar leaves. However,auracyanin appears to have sequence characteristics of both small copperprotein sequence classes. The overall similarity with a consensussequence of azurin is roughly the same as that with a consensus sequenceof plastocyanin, namely 30.5%. The N-terminal sequence region 1-18 ofauracyanin is remarkably rich in glycine and hydroxy amino acids. Id.See exemplary amino acid sequence SEQ ID NO: 16 for chain A ofauracyanin from Chloroflexus aurantiacus (NCBI Protein Data BankAccession No. AAM12874).

The auracyanin B molecule has a standard cupredoxin fold. The crystalstructure of auracyanin B from Chloroflexus aurantiacus has beenstudied. (Bond et al., J. Mol. Biol. 306:47-67 (2001).) With theexception of an additional N-terminal strand, the molecule is verysimilar to that of the bacterial cupredoxin, azurin. As in othercupredoxins, one of the Cu ligands lies on strand 4 of the polypeptide,and the other three lie along a large loop between strands 7 and 8. TheCu site geometry is discussed with reference to the amino acid spacingbetween the latter three ligands. The crystallographically characterizedCu-binding domain of auracyanin B is probably tethered to theperiplasmic side of the cytoplasmic membrane by an N-terminal tail thatexhibits significant sequence identity with known tethers in severalother membrane-associated electron-transfer proteins. The amino acidsequences of the B forms are presented in McManus et al. (J. Biol. Chem.267:6531-6540 (1992).). See exemplary amino acid sequence SEQ ID NO: 17for chain B of auracyanin from Chloroflexus aurantiacus (NCBI ProteinData Bank Accession No. 1QHQA).

Stellacyanin

Stellacyanins are a subclass of phytocyanins, a ubiquitous family ofplant cupredoxins. An exemplary sequence of a stellacyanin is includedherein as SEQ ID NO: 15. The crystal structure of umecyanin, astellacyanin from horseradish root (Koch et al., J. Am. Chem. Soc.127:158-166 (2005)) and cucumber stellacyanin (Hart et al., ProteinScience 5:2175-2183 (1996).). The protein has an overall fold similar tothe other phytocyanins. The ephrin B2 protein ectodomain tertiarystructure bears a significant similarity to stellacyanin. (Toth et al.,Developmental Cell 1:83-92 (2001).) An exemplary amino acid sequence ofa stellacyanin is found in the National Center for BiotechnologyInformation Protein Data Bank as Accession No. 1JER, SEQ ID NO: 15.

Cucumber Basic Protein

An exemplary amino acid sequence from a cucumber basic protein isincluded herein as SEQ ID NO: 18. The crystal structure of the cucumberbasic protein (CBP), a type 1 blue copper protein, has been refined at1.8 Å resolution. The molecule resembles other blue copper proteins inhaving a Greek key beta-barrel structure, except that the barrel is openon one side and is better described as a “beta-sandwich” or “beta-taco”.(Guss et al., J. Mol. Biol. 262:686-705 (1996).) The ephrinB2 proteinectodomain tertiary structure bears a high similarity (rms deviation 1.5Å for the 50α carbons) to the cucumber basic protein. (Toth et al.,Developmental Cell 1:83-92 (2001).)

The Cu atom has the normal blue copper NNSS′ co-ordination with bondlengths Cu—N(His39)=1.93 A, Cu—S(Cys79)=2.16 A, Cu—N(His84)=1.95 A,Cu—S(Met89)=2.61 A. A disulphide link (Cys52)S—S-(Cys85), appears toplay an important role in stabilizing the molecular structure. Thepolypeptide fold is typical of a sub-family of blue copper proteins(phytocyanins) as well as a non-metalloprotein, ragweed allergen Ra3,with which CBP has a high degree of sequence identity. The proteinscurrently identifiable as phytocyanins are CBP, stellacyanin,mavicyanin, umecyanin, a cucumber peeling cupredoxin, a putative bluecopper protein in pea pods, and a blue copper protein from Arabidopsisthaliana. In all except CBP and the pea-pod protein, the axialmethionine ligand normally found at blue copper sites is replaced byglutamine. An exemplary sequence for cucumber basic protein is found inNCBI Protein Data Bank Accession No. 2CBP, SEQ ID NO: 18.

Cytochromes

Cytochrome C₅₅₁

Cytochrome C₅₅₁ from P. aeruginosa (Pa-C551) is a monomeric redoxprotein of 82 amino-acid residues (SEQ ID NO: 2), involved indissimilative denitrification as the physiological electron donor ofnitrite reductase. The functional properties of Pa-C551 have beenextensively investigated. The reactions with non-physiological smallinorganic redox reactants and with other macromolecules, like bluecopper proteins, eukaryotic cytochrome c and the physiological partnernitrite reductase have provided a test for protein-protein electrontransfer.

The three-dimensional structure of Pa-C551, which is a member ofbacterial class I cytochromes, shows a single low-spin heme with His-Metligation and the typical polypeptide fold which however leaves the edgesof pyrrole rings II and III of the heme exposed (Cutruzzola et al., J.Inorgan. Chem., 88:353-61 (2002)). The lack of a 20-residue omega loop,present in the mammalian class I cytochromes, causes further exposure ofthe heme edge at the level of propionate 13. The distribution of chargedresidues on the surface of Pa-C551 is very anisotropic: one side isricher in acidic residues whereas the other displays a ring of positiveside chains, mainly lysines, located at the border of a hydrophobicpatch which surrounds the heme crevice. This patch comprises residuesGly11, Val13, Ala14, Met22, Val23, Pro58, Ile59, Pro60, Pro62, Pro63 andAla65. The anisotropic charge distribution leads to a large dipolarmoment which is important for electron transfer complex formation.

The charge distribution described above for Pa-C551 has been reportedfor other electron transfer proteins and their electron acceptors.Moreover, modification by site-directed mutagenesis of residues withinthe hydrophobic or charged patch has shown for different proteins theimportance of surface complementarity for binding and electron transfer.As an example, evidence for the relevance of the hydrophobic patch forthe electron transfer properties of azurin from P. aeruginosa came fromthe studies carried out on mutants of residues Met44 and Met64 changedto positively and negatively charged amino acids. Id.

The cytochrome c-type domain has a fold consisting of a series of alphahelices and reverse turns that serve to envelop the covalently boundhaem within a hydrophobic pocket. This domain can be found in monodomaincytochrome c proteins, such as cytochrome c6, cytochrome c₅₅₂,cytochrome c₄₉₅ and mitochondrial cytochrome c. The cytochrome c-typedomain occurs in a number of other proteins, such as in cytochromecd1-nitrite reductase as the N-terminal haem c domain, in quinoproteinalcohol dehydrogenase as the C-terminal domain, in Quinohemoproteinamine dehydrogenase A chain as domains 1 and 2, and in the cytochromebc₁ complex as the cytochrome bc₁ domain. Structural analysis with VASTalgorithm (cytochrome c₅₅₁ from Pseudomonas aeruginosa as a query)showed significant structural neighbors (P values between 10^(−10.3) to10^(−4.5)) only for cytochromes.

Methods of Use

The invention provides methods to treat patients with a malarialinfection or at danger of acquiring one, or inhibit the spread of themalaria parasite. These methods comprise administering to a patient oran insect vector a cupredoxin or cytochrome, or variant, derivative orstructural equivalent thereof which inhibits parasitemia ofmalaria-infected mammalian cells. The inhibition of parasitemia can bedetermined by many methods well known in the art. One method isdescribed in Example 6, and determines the inhibition of parasitemia inmalaria-infected human red blood cells. In other embodiments, thecupredoxin or cytochrome, or variant, derivative or structuralequivalent thereof inhibits intracellular replication of the malariaparasite in human red blood cells and is administered to the patient orinsect vector. Methods to determine the intracellular replication of themalaria parasite are well known in the art, and one such method isdescribed in Example 2. The mode of the invention is not limited to anyparticular mechanism, and inhibition of parasitemia may result from manyfactors, including but not limited to, inhibition of replication of theparasite in infected blood cells, inhibition of parasite infectinguninfected blood cells, inhibition in the growth cycle of the parasiteand inhibition of parasite entry into the mammalian cell.

The invention provides methods to treat patients suffering frominfection by a malaria parasite by administering an effective amount ofat least one protein that is a cupredoxin or cytochrome, or variant,derivative or structural equivalent thereof. The patients that may betreated by this method are any mammal that can be infected by a malariaparasite, and specifically are human patients. Malaria parasites knownto infect mammals include, but are not limited to, Plasmodiumfalciparum, P. vivax, P. berghei (rodent-specific), P. yoelli(murine-specific), P. cynomolgi and P. knowlesi (monkey-specific).

It has also been learned that cupredoxins and cytochrome c₅₅₁ are alsoeffective against HIV-I infections, as disclosed in a co-filedapplication. Methods For Treating HIV Infection With Cupredoxin AndCytochrome C,” U.S. Provisional Patent Application Ser. No. 60/682,833,whose disclosure is expressly incorporated herein by reference. Further,co-infections with HIV and malaria are very common in many areas of theworld, and in particular sub-Saharan Africa. In some embodiments, thepatient suffering from infection by a malaria parasite is also sufferingfrom infection by HIV. In some embodiments, the method of treatment ofthe invention also comprises administering anti-HIV drugs. In someembodiments, the anti-HIV drugs are co-administered.

The invention also provides methods to treat a patient suspected ofhaving contact with a malaria parasite by administering an effectiveamount of at least one peptide that is a cupredoxin and/or a cytochrome,or a variants derivative or structural equivalent of a cupredoxin or acytochrome. A patient can be suspected of having contact with a malariaparasite, for example, if that patient lives or has traveled in a regionof the world where malaria infection of others of the patient's speciesis common. Treatment by this method may be commenced when the patient isabout to, or has already, come into contact with the malaria parasite.Contact with malaria parasites most often occurs by contact with aninsect vector such as mosquitoes, so that areas abundant in theseinsects and the malaria parasite are considered to be among the areaswhere a patient would have a high probability of coming in contact witha malaria parasite. Such areas of the world include, but are not limitedto, parts of Africa, Asia and Latin America. Further, a patient can besuspected of having contact with the malaria parasite if they have comeinto contact with blood infected with a malaria parasite, areintentionally exposed to the malaria parasite, or accidentally injectedwith blood or drugs contaminated with the parasite.

The cupredoxin or cytochrome, or variant, derivative or structuralequivalent of cupredoxin or cytochrome can be administered to thepatient by many routes and in many regimens that will be well known tothose in the art. In specific embodiments, the cupredoxin or cytochrome,or variant, derivative or structural equivalent of cupredoxin orcytochrome is administered orally, topically, by inhalation, byinjection, more specifically, intravenously, intramuscularly orsubcutaneously.

In one embodiment, the methods may comprise co-administering to apatient one unit dose of compositions comprising a cupredoxin orcytochrome, or a variant, derivative or structural equivalent thereofand one unit dose of compositions comprising an anti-malarial drugand/or an anti-HIV drug, in either order. These compositions may beadministered at about the same time, or within about a given timefollowing the administration of the other, for example, about one minuteto about 60 minutes, or about 1 hour to about 12 hours of the other.

The invention also provides methods to inhibit the spread of the malariaparasite in an insect vector population harboring a malaria parasite byadministering to an insect vector in the population at least one of acupredoxin or cytochrome, or variant, derivative or structuralequivalent of cupredoxin or cytochrome, at an amount that is effectiveto reduce the infectivity of the parasite in a co-existant mammalianpopulation. In specific embodiments, the insect vector is a mosquito,and more specifically a mosquito from the species Anopheles gambiae. Inthis method, the administration of the cupredoxin or cytochrome, orvariant, derivative or structural equivalent of cupredoxin or cytochromecan be accomplished by placing the peptides in compositions that will beconsumed by the insect vector, however any manner that brings thepeptide into contact with the malaria parasite in the insect vector'sgut is contemplated. Many methods to administer chemicals to insectpopulations which produce such consumption are known in the art.

In another embodiment, a transmissible genetic element that passes fromone mosquito to another will be operably connected to the cupredoxincoding sequence operably connected to a constitutive promoter, thecupredoxin or cytochrome, or variant, derivative or structuralequivalent of cupredoxin or cytochrome will be produced inside theAnopheles gambiae infected with P. falciparum and will interfere withits replication/survival in the mosquito.

Other manners of administration of the peptides to the insect vectorinclude, but are not limited to, fusing the cupredoxin or cytochrome, orvariant, derivative or structural equivalent of cupredoxin or cytochromegenes to genes from other proteins normally consumed by the insects. Theamount of peptides administered to the insect vector should be an amounteffective to reduce the infectivity of the malaria parasite in a mammalwhen the insect vector comes into contact with a mammal. In specificembodiments, the amount administered should be effective to reduce theinfectivity of the malaria parasite when the insect vector comes intocontact with a human.

Mosquito larvae are suitable for use in the present invention andpreferably, the promoter used is a strong promoter. Two alternativecategories of promoter are available for use: inducible and constitutivepromoters. Inducible promoters include, for example, heat shockpromoters. Preferably, the heat shock promoter is an insect heat shockpromoter, for example the Drosophila melanogaster hsp70 promoter, whichis capable of driving the expression of genes in heterologous organisms,including medfly. The invention also encompasses the use of the medflyhsp70 promoter (Papadimitriou et al., Insect Mol Biol 7:279-90 (1998)).Alternative systems may be based on induction with the antibiotictetracycline. (Heinrich and Scott, PNAS 97:8229-8232, (2000))

Heat shock promoters are inducible by raising the temperature of theconditions under which the medfly are being cultured. For example, at23-25° C., the hsp70 promoter is active at low levels or not at all.This allows the insect larva to develop without stress induced by theproduction of a heterologous protein. At higher temperatures, however,such as 37-42° C., the hsp70 promoter is induced and expresses theheterologous protein at a high level.

Inducible promoters may be constructed based on known inducible genecontrol elements. For example, inducible promoters may be constructed bycombining an element responsive to a drug or hormone which may beadministered in the diet. In a preferred embodiment, a human oestrogenresponsive element (ERE) may be used to regulate expression of theprotein of interest, as long as the insect is transformed with a secondcoding sequence which expresses the human oestrogen receptor.

Constitutive promoters may also be used to express the protein and/orother proteins required in the insect larva. For example, theconstitutive promoter may be a cytoplasmic actin promoter. The D.melanogaster cytoplasmic actin promoter has been cloned (Act5C) and ishighly active in mosquitoes (Huynh and Zieler, J. Mol. Biol. 288:13-20(1999)). Cytoplasmic actin genes and their promoters may also beisolated from other insects, including medfly. Other examples includethe cytoplasmic tubulin promoter, for instance the medfly cytoplasmictubulin promoter.

Promoters which control secreted polypeptides may be used, optionallytogether with appropriate signal sequences, to direct secretion of theprotein to the haemolymph. For example, the larval serum proteinpromoter may be employed (Benes et al., Mol. Gen. Genet. 249(5):545-556(1995)).

Mass rearing technology for mosquitoes is highly developed. For proteinproduction, larval cultures that have been initiated at different timescan be synchronized by appropriate temperature shift regimes. This ispossible because growth rates depend on the temperature of theenvironment; at 18° C. larval growth rates decrease by approximately50%.

Pharmaceutical Compositions Comprising Cupredoxin and/or Cytochrome andVariants and Derivatives Thereof

Pharmaceutical compositions comprising cupredoxin or cytochrome, orvariant, derivative or structural equivalent thereof can be manufacturedin any conventional manner, e.g., by conventional mixing, dissolving,granulating, dragee-making, emulsifying, encapsulating, entrapping, orlyophilizing processes. The substantially pure cupredoxin and/orcytochrome, and variants, derivatives and structural equivalents thereofcan be readily combined with a pharmaceutically acceptable carrierwell-known in the art. Such carriers enable the preparation to beformulated as a tablet, pill, dragee, capsule, liquid, gel, syrup,slurry, suspension, and the like. Suitable carriers or excipients canalso include, for example, fillers and cellulose preparations. Otherexcipients can include, for example, flavoring agents, coloring agents,detackifiers, thickeners, and other acceptable additives, adjuvants, orbinders. In some embodiments, the pharmaceutical preparation issubstantially free of preservatives. In other embodiments, thepharmaceutical preparation may contain at least one preservative.General methodology on pharmaceutical dosage forms is found in Ansel etal., Pharmaceutical Dosage Forr's and Drug Delivery Systems (LippencottWilliams & Wilkins, Baltimore Md. (1999)).

The composition comprising a cupredoxin or cytochrome, or variant,derivative or structural equivalent thereof used in the invention may beadministered in a variety of ways, including by injection (e.g.,intradermal, subcutaneous, intramuscular, intraperitoneal and the like),by inhalation, by topical administration, by suppository, by using atransdermal patch or by mouth. General information on drug deliverysystems can be found in Ansel et al., Id. In some embodiments, thecomposition comprising a cupredoxin or cytochrome, or variant,derivative or structural equivalent thereof can be formulated and useddirectly as injectibles, for subcutaneous and intravenous injection,among others. The injectable formulation, in particular, canadvantageously be used to treat patients that are at risk of anmalaria-infection, likely to have an malaria-infection or have anmalaria-infection. The composition comprising a cupredoxin orcytochrome, or variant, derivative or structural equivalent thereof canalso be taken orally after mixing with protective agents such aspolypropylene glycols or similar coating agents.

When administration is by injection, the cupredoxin or cytochrome, orvariant, derivative or structural equivalent thereof may be formulatedin aqueous solutions, specifically in physiologically compatible bufferssuch as Hanks solution, Ringer's solution, or physiological salinebuffer. The solution may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the cupredoxin orcytochrome, or variant, derivative or structural equivalent thereof maybe in powder form for constitution with a suitable vehicle, e.g.,sterile pyrogen-free water, before use. In some embodiments, thepharmaceutical composition does not comprise an adjuvant or any othersubstance added to enhance the immune response stimulated by thepeptide. In some embodiments, the pharmaceutical composition comprises asubstance that inhibits an immune response to the peptide.

When administration is by intravenous fluids, the intravenous fluids foruse administering the cupredoxin or cytochrome, or variant, derivativeor structural equivalent thereof may be composed of crystalloids orcolloids. Crystalloids as used herein are aqueous solutions of mineralsalts or other water-soluble molecules. Colloids as used herein containlarger insoluble molecules, such as gelatin. Intravenous fluids may besterile.

Crystalloid fluids that may be used for intravenous administrationinclude but are not limited to, normal saline (a solution of sodiumchloride at 0.9% concentration), Ringer's lactate or Ringer's solution,and a solution of 5% dextrose in water sometimes called D5W, asdescribed in Table 2.

TABLE 2 Composition of Common Crystalloid Solutions Solution Other Name[Na⁺] [Cl⁻] [Glucose] D5W 5% Dextrose 0 0 252 ⅔ & ⅓ 3.3% Dextrose/ 51 51168 0.3% saline Half-normal 0.45% NaCl 77 77 0 saline Normal saline 0.9%NaCl 154 154 0 Ringer's Ringer's 130 109 0 lactate* solution *Ringer'slactate also has 28 mmol/L lactate, 4 mmol/L K⁺ and 3 mmol/L Ca²⁺.

When administration is by inhalation, the cupredoxin or cytochrome, orvariant, derivative or structural equivalent thereof may be delivered inthe form of an aerosol spray from pressurized packs or a nebulizer withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin, for use in an inhaler or insufflator may beformulated containing a powder mix of the proteins and a suitable powderbase such as lactose or starch.

When administration is by topical administration, the cupredoxin orcytochrome, or variant, derivative or structural equivalent thereof maybe formulated as solutions, gels, ointments, creams, suspensions, andthe like, as are well known in the art. In some embodiments,administration is by means of a transdermal patch. When administrationis by suppository (e.g., rectal or vaginal), cupredoxin and/orcytochrome c and variants and derivatives thereof compositions may alsobe formulated in compositions containing conventional suppository bases.

When administration is oral, a cupredoxin or cytochrome, or variant,derivative or structural equivalent thereof can be readily formulated bycombining the cupredoxin or cytochrome, or variant, derivative orstructural equivalent thereof with pharmaceutically acceptable carrierswell known in the art. A solid carrier, such as mannitol, lactose,magnesium stearate, and the like may be employed; such carriers enablethe cupredoxin and/or cytochrome and variants and derivatives thereof tobe formulated as tablets, pills, dragees, capsules, liquids, gels,syrups, slurries, suspensions and the like, for oral ingestion by asubject to be treated. For oral solid formulations such as, for example,powders, capsules and tablets, suitable excipients include fillers suchas sugars, cellulose preparation, granulating agents, and bindingagents.

Other convenient carriers, as well-known in the art, also includemultivalent carriers, such as bacterial capsular polysaccharide, adextran or a genetically engineered vector. In addition,sustained-release formulations that include a cupredoxin or cytochrome,or variant, derivative or structural equivalent thereof allow for therelease of cupredoxin or cytochrome, or variant, derivative orstructural equivalent thereof over extended periods of time, such thatwithout the sustained release formulation, the cupredoxin or cytochrome,or variant, derivative or structural equivalent thereof would be clearedfrom a subject's system, and/or degraded by, for example, proteases andsimple hydrolysis before eliciting or enhancing a therapeutic effect.

The half-life in the bloodstream of the compositions of the inventioncan be extended or optimized by several methods well known to those inthe art including but not limited to, circularized peptides (Monk etal., BioDrugs 19(4):261-78, (2005); DeFreest et al., J. Pept. Res.63(5):409-19 (2004)), D,L-peptides (diastereomer), (Futaki et al., J.Biol. Chem. Feb. 23; 276(8):5836-40 (2001); Papo et al., Cancer Res.64(16):5779-86 (2004); Miller et al., Biochem. Pharmacol. 36(1):169-76,(1987)); peptides containing unusual amino acids (Lee et al., J. Pept.Res. 63(2):69-84 (2004)), N- and C-terminal modifications (Labrie etal., Clin. Invest. Med. 13(5):275-8, (1990)), and hydrocarbon stapling(Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000); Walenskiet al., Science 305:1466-1470 (2004)). Of particular interest ared-isomerization (substitution) and modification of peptide stability viaD-substitution or L-amino acid substitution and hydrocarbon stapling.

In various embodiments, the pharmaceutical composition includes carriersand excipients (including but not limited to buffers, carbohydrates,mannitol, proteins, polypeptides or amino acids such as glycine,antioxidants, bacteriostats, chelating agents, suspending agents,thickening agents and/or preservatives), water, oils, saline solutions,aqueous dextrose and glycerol solutions, other pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such as buffering agents, tonicity adjusting agents, wettingagents and the like. It will be recognized that, while any suitablecarrier known to those of ordinary skill in the art may be employed toadminister the compositions of this invention, the type of carrier willvary depending on the mode of administration. Compounds may also beencapsulated within liposomes using well-known technology. Biodegradablemicrospheres may also be employed as carriers for the pharmaceuticalcompositions of this invention. Suitable biodegradable microspheres aredisclosed, for example, in U.S. Pat. Nos. 4,897,268; 5,075,109;5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344 and 5,942,252.

The pharmaceutical compositions may be sterilized by conventional,well-known sterilization techniques, or may be sterile filtered. Theresulting aqueous solutions may be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterilesolution prior to administration.

Administration of Cupredoxin and/or Cytochrome and Variants andDerivatives Thereof

The cupredoxin or cytochrome, or variant, derivative or structuralequivalent thereof can be administered formulated as pharmaceuticalcompositions and administered by any suitable route, for example, byoral, buccal, inhalation, sublingual, rectal, vaginal, transurethral,nasal, topical, percutaneous, i.e., transdermal or parenteral (includingintravenous, intramuscular, subcutaneous and intracoronary)administration. The pharmaceutical formulations thereof can beadministered in any amount effective to achieve its intended purpose.More specifically, the composition is administered in a therapeuticallyeffective amount. In specific embodiments, the therapeutically effectiveamount is generally from about 0.01-20 mg/day 1 kg of body weight.

The compounds comprising cupredoxin or cytochrome, or variant,derivative or structural equivalent thereof are useful for the treatmentand/or prophylaxis of malaria infection, alone or in combination withother active agents. The appropriate dosage will, of course, varydepending upon, for example, the compound of cupredoxin or cytochrome,or variant, derivative or structural equivalent thereof employed, thehost, the mode of administration and the nature and severity of theconditions being treated. However, in general, satisfactory results inhumans are indicated to be obtained at daily dosages from about 0.01-20mg/kg of body weight. An indicated daily dosage in humans is in therange from about 0.7 mg to about 1400 mg of a compound of cupredoxin orcytochrome c₅₅₁, or variant, derivative or structural equivalent thereofconveniently administered, for example, in daily doses, weekly doses,monthly doses, and/or continuous dosing. Daily doses can be in discretedosages from 1 to 12 times per day. Alternatively, doses can beadministered every other day, every third day, every fourth day, everyfifth day, every sixth day, every week, and similarly in day incrementsup to 31 days. Alternatively, dosing can be continuous using patches,i.v. administration and the like.

The method of introducing cupredoxin or cytochrome, or variant,derivative or structural equivalent thereof to patients is, in someembodiments, through the co-administration of cupredoxin or cytochrome,or variant, derivative or structural equivalent thereof with other drugsused for malaria therapy. Such methods are well-known in the art. In aspecific embodiment, the cupredoxin and/or cytochrome c are part of ancocktail or co-dosing containing or with other malaria therapeutics.Malaria therapeutics of interest include, but are not limited to,proguanil, chlorproguanil, trimethoprim, chloroquine, mefloquine,lumefantrine, atovaquone, pyrimethamine-sulfadoxine,pyrimethamine-dapsone, halofantrine, quinine, quinidine, amodiaquine,amopyroquine, sulphonamides, artemisinin, arteflene, artemether,artesunate, primaquine, pyronaridine, proguanil, chloroquine,mefloquine, pyrimethamine-sulfadoxine, pyrimethamine-dapsone,halofantrine, quinine, proguanil, chloroquine, mefloquine,1,16-hexadecamethylenebis(N-methylpyrrolidinium)dibromide, andcombinations thereof.

The method of introducing cupredoxin or cytochrome c₅₅₁, or variant,derivative or structural equivalent thereof to patients is, in someembodiments, the same as currently used to introduce anti-HIV drugs,such as the protease-inhibitor-containing cocktails. Such methods arewell-known in the art. In a specific embodiment, the cupredoxin orcytochrome c₅₅₁, or variant, derivative or structural equivalent thereofare part of an cocktail or co-dosing with anti-HIV therapeutics.Anti-HIV drugs include, but are not limited to, reverse transcriptaseinhibitors: AZT (zidovudine [Retrovir]), ddC (zalcitabine [Hivid],dideoxyinosine), d4T (stavudine [Zerit]), and 3TC (lamivudine [Epivir]),nonnucleoside reverse transcriptase inhibitors (NNRTIS): delavirdine(Rescriptor) and nevirapine (Viramune), protease inhibitors: ritonavir(Norvir), a lopinavir and ritonavir combination (Kaletra), saquinavir(Invirase), indinavir sulphate (Crixivan), amprenavir (Agenerase), andnelfinavir (Viracept). In some embodiments, a combination of severaldrugs called highly active antiretroviral therapy (HAART) is used totreat the patients.

The exact formulation, route of administration, and dosage is determinedby the attending physician in view of the patient's condition. Dosageamount and interval can be adjusted individually to provide plasmalevels of the active cupredoxin or cytochrome, or variant, derivative orstructural equivalent thereof which are sufficient to maintaintherapeutic effect. Generally, the desired cupredoxin or cytochrome, orvariant, derivative or structural equivalent thereof is administered inan admixture with a pharmaceutical carrier selected with regard to theintended route of administration and standard pharmaceutical practice.

In one aspect, the cupredoxin or cytochrome, or variant, derivative orstructural equivalent thereof is delivered as DNA such that thepolypeptide is generated in situ. In one embodiment, the DNA is “naked,”as described, for example, in Ulmer et al., (Science 259:1745-1749(1993)) and reviewed by Cohen (Science 259:1691-1692 (1993)). The uptakeof naked DNA may be increased by coating the DNA onto a carrier, e.g.,biodegradable beads, which are then efficiently transported into thecells. In such methods, the DNA may be present within any of a varietyof delivery systems known to those of ordinary skill in the art,including nucleic acid expression systems, bacterial and viralexpression systems. Techniques for incorporating DNA into suchexpression systems are well known to those of ordinary skill in the art.See, e.g., WO90/11092, WO93/24640, WO 93/17706, and U.S. Pat. No.5,736,524.

Vectors, used to shuttle genetic material from organism to organism, canbe divided into two general classes: cloning vectors are replicatingplasmid or phage with regions that are essential for propagation in anappropriate host cell and into which foreign DNA can be inserted; theforeign DNA is replicated and propagated as if it were a component ofthe vector. An expression vector (such as a plasmid, yeast, or animalvirus genome) is used to introduce foreign genetic material into a hostcell or tissue in order to transcribe and translate the foreign DNA,such as the DNA of a cupredoxin and/or a cytochrome. In expressionvectors, the introduced DNA is operably-linked to elements such aspromoters that signal to the host cell to highly transcribe the insertedDNA. Some promoters are exceptionally useful, such as induciblepromoters that control gene transcription in response to specificfactors Operably-linking a cupredoxin or cytochrome and variants andderivatives thereof polynucleotide to an inducible promoter can controlthe expression of the cupredoxin or cytochrome and variants andderivatives thereof in response to specific factors. Examples of classicinducible promoters include those that are responsive to α-interferon,heat shock, heavy metal ions, and steroids such as glucocorticoids(Kaufman, Methods Enzymol. 185:487-511 (1990)) and tetracycline. Otherdesirable inducible promoters include those that are not endogenous tothe cells in which the construct is being introduced, but, however, areresponsive in those cells when the induction agent is exogenouslysupplied. In general, useful expression vectors are often plasmids.However, other forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses) are contemplated.

Vector choice is dictated by the organism or cells being used and thedesired fate of the vector. In general, vectors comprise signalsequences, origins of replication, marker genes, polylinker sites,enhancer elements, promoters, and transcription termination sequences.As an example, one may clone a cupredoxin or a cytochrome gene into avector transmissible in the malaria parasite-harboring mosquitoes toprevent the parasite from replicating inside the mosquitoes. Thetransmissibility of the vector will allow the spread of thecupredoxin/cytochrome to neighboring mosquitoes that are infected withthe malaria parasites as well.

The exact formulation, route of administration, and dosage is determinedby the attending physician in view of the patient's condition. Dosageamount and interval can be adjusted individually to provide plasmalevels of the active cupredoxin and/or cytochrome and variants andderivatives thereof which are sufficient to treat the patient and/ormaintain therapeutic effect. Generally, the desired cupredoxin and/orcytochrome and variants and derivatives thereof can be administered inan admixture with a pharmaceutical carrier selected with regard to theintended route of administration and standard pharmaceutical practice.Pharmaceutical compositions used in accordance with the presentinvention can be formulated in a conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries that facilitate processing of the cupredoxin and/orcytochrome and variants and derivatives thereof, active agents, forinhibiting or stimulating the secretion of cupredoxin and/or cytochromeand variants and derivatives thereof, or a mixture thereof intopreparations which can be used therapeutically.

Kits Comprising Cupredoxin and/or Cytochrome C And Variants andDerivatives Thereof

In one aspect, the invention provides kits containing one or more of thefollowing in a package or container: (1) a biologically activecomposition comprising a cupredoxin or cytochrome, or variant,derivative or structural equivalent thereof; (2) a pharmaceuticallyacceptable adjuvant or excipient; (3) a vehicle for administration, suchas a syringe; (4) instructions for administration. Embodiments in whichtwo or more of components (1)-(4) are found in the same container arealso contemplated.

In another aspect, the invention provides kits containing one or more ofthe following in a package or container: (1) a biologically activecomposition comprising a cupredoxin or cytochrome, or variant,derivative or structural equivalent thereof; (2) an malaria therapeutic,including, but not limited to, proguanil, chlorproguanil, trimethoprim,chloroquine, mefloquine, lumefantrine, atovaquone,pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halofantrine, quinine,quinidine, amodiaquine, amopyroquine, sulphonamides, artemisinin,arteflene, artemether, artesunate, primaquine, pyronaridine, proguanil,chloroquine, mefloquine, pyrimethamine-sulfadoxine,pyrimethamine-dapsone, halofantrine, quinine, proguanil, chloroquine,mefloquine, 1,16-hexadecamethylenebis(N-methylpyrrolidinium)dibromide;(3) a pharmaceutically acceptable adjuvant or excipient; (4) a vehiclefor administration, such as a syringe; (5) instructions foradministration. Embodiments in which two or more of components (1)-(5)are found in the same packaging or container are also contemplated.

In some embodiments, the kit also comprises an anti-HIV therapeutic in apackage or container. Anti-HIV therapeutics of interest include, but arenot limited to, reverse transcriptase inhibitors: AZT (zidovudine[Retrovir]), ddC (zalcitabine [Hivid], dideoxyinosine), d4T (stavudine[Zerit]), and 3TC (lamivudine [Epivir]), nonnucleoside reversetranscriptase inhibitors (NNRTIS): delavirdine (Rescriptor) andnevirapine (Viramune), protease inhibitors: ritonavir (Norvir), alopinavir and ritonavir combination (Kaletra), saquinavir (Invirase),indinavir sulphate (Crixivan), amprenavir (Agenerase), and nelfinavir(Viracept). In some embodiment, a combination of several drugs calledhighly active antiretroviral therapy (HAART) is included in the kit.

When a kit is supplied, the different components of the composition maybe packaged in separate containers and admixed immediately before use.Such packaging of the components separately may permit long-term storagewithout losing the active components' functions.

The reagents included in the kits can be supplied in containers of anysort such that the life of the different components are preserved andare not adsorbed or altered by the materials of the container. Forexample, sealed glass ampules may contain the lyophilized polypeptide orpolynucleotide of cupredoxin and/or cytochrome c and variants andderivatives thereof, or buffers that have been packaged under a neutral,non-reacting gas, such as nitrogen. Ampules may consist of any suitablematerial, such as glass, organic polymers, such as polycarbonate,polystyrene, etc., ceramic, metal or any other material typicallyemployed to hold similar reagents. Other examples of suitable containersinclude simple bottles that may be fabricated from similar substances asampules, and envelopes, that may comprise foil-lined interiors, such asaluminum or an alloy. Other containers include test tubes, vials,flasks, bottles, syringes, or the like Containers may have a sterileaccess port, such as a bottle having a stopper that can be pierced by ahypodermic injection needle. Other containers may have two compartmentsthat are separated by a readily removable membrane that upon removalpermits the components to be mixed. Removable membranes may be glass,plastic, rubber, etc.

Kits may also be supplied with instructional materials. Instructions maybe printed on paper or other substrate, and/or may be supplied as anelectronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zipdisc, videotape, audiotape, flash memory device, etc. Detailedinstructions may not be physically associated with the kit; instead, auser may be directed to an internet web site specified by themanufacturer or distributor of the kit, or supplied as electronic mail.

Modification of Cupredoxin and/or Cytochrome

Cupredoxin or cytochrome may be chemically modified or geneticallyaltered to produce variants and derivatives as explained above. Suchvariants and derivatives may be synthesized by standard techniques.

In addition to naturally-occurring allelic variants of cupredoxin andcytochrome, changes can be introduced by mutation into cupredoxin orcytochrome coding sequence that incur alterations in the amino acidsequences of the encoded cupredoxin or cytochrome that do notsignificantly alter the ability of cupredoxin or cytochrome to inhibitparasitemia in malaria-infected red blood cells. A “non-essential” aminoacid residue is a residue that can be altered from the wild-typesequences of the cupredoxin without altering biological activity,whereas an “essential” amino acid residue is required for suchbiological activity. For example, amino acid residues that are conservedamong the cupredoxins are predicted to be particularly non-amenable toalteration, and thus “essential.”

Amino acids for which conservative substitutions that do not change theactivity of the polypeptide can be made are well known in the art.Useful conservative substitutions are shown in Table 3, “Preferredsubstitutions.” Conservative substitutions whereby an amino acid of oneclass is replaced with another amino acid of the same type fall withinthe scope of the invention so long as the substitution does notmaterially alter the biological activity of the compound.

TABLE 3 Preferred substitutions Preferred Original residue Exemplarysubstitutions substitutions Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln,Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) Ser SerGln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro, Ala Ala His (H) Asn, Gln,Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu Norleucine Leu (L)Norleucine, Ile, Val, Met, Ala, Ile Phe Lys (K) Arg, Gln, Asn Arg Met(M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) AlaAla Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp,Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Ala, Leu Norleucine

Non-conservative substitutions that affect (1) the structure of thepolypeptide backbone, such as a β-sheet or α-helical conformation, (2)the charge, (3) hydrophobicity, or (4) the bulk of the side chain of thetarget site can modify the cytotoxic factor function. Residues aredivided into groups based on common side-chain properties as denoted inTable 4. Non-conservative substitutions entail exchanging a member ofone of these classes for another class. Substitutions may be introducedinto conservative substitution sites or more specifically intonon-conserved sites.

TABLE 4 Amino acid classes Class Amino acids hydrophobic Norleucine,Met, Ala, Val, Leu, Ile neutral hydrophilic Cys, Ser, Thr acidic Asp,Glu basic Asn, Gln, His, Lys, Arg disrupt chain conformation Gly, Proaromatic Trp, Tyr, Phe

The variant polypeptides can be made using methods known in the art suchas oligonucleotide-mediated (site-directed) mutagenesis, alaninescanning, and PCR mutagenesis. Site-directed mutagenesis (Carter,Biochem J. 237:1-7 (1986); Zoller and Smith, Methods Enzymol.154:329-350 (1987)), cassette mutagenesis, restriction selectionmutagenesis (Wells et al., Gene 34:315-323 (1985)) or other knowntechniques can be performed on the cloned DNA to produce the cupredoxinor cytochrome c₅₅₁ variant DNA.

Known mutations of cupredoxins and cytochrome c₅₅₁ can also be used tocreate variant cupredoxin and cytochrome c₅₅₁ to be used in the methodsof the invention. For example, the C112D and M44KM64E mutants of azurinare known to have cytotoxic and growth arresting activity that isdifferent from the native azurin, and such altered activity can beuseful in the treatment methods of the present invention. One embodimentof the methods of the invention utilize cupredoxin and/or cytochrome andvariants and derivatives thereof retaining the ability inhibit thegrowth of malaria infection in mammalian cells. In another embodiment,the methods of the present invention utilize cupredoxin variants such asthe M44KM64E mutant, having the ability to cause cellular growth arrest.

A more complete understanding of the present invention can be obtainedby reference to the following specific Examples. The Examples aredescribed solely for purposes of illustration and are not intended tolimit the scope of the invention, Changes in form and substitution ofequivalents are contemplated as circumstances may suggest or renderexpedient. Although specific terms have been employed herein, such termsare intended in a descriptive sense and not for purposes of limitations.Modifications and variations of the invention as hereinbefore set forthcan be made without departing from the spirit and scope thereof, and,therefore, only such limitations should be imposed as are indicated bythe appended embodiments.

EXAMPLES Example 1 In Vitro Inhibition of P. falciparum Parasitemia byCupredoxin and Cytochrome

The cupredoxins bacterial wt azurin, M44KM64E azurin, rusticyanin andcyanobacterial plastocyanin, as well as the cytochromes Pseudomonasaeruginosa cytochrome c₅₅₁, human cytochrome c and Phormidium laminosumcytochrome f were tested in a normal red blood cell (RBC) assay at 200μg/ml concentrations at 30 hours post inoculation. In these experiments,the normal RBCs were washed twice in serum free media and resuspended to10% hematocrit in complete RPMI. 200 μl of 10% Hct RBCs were added toeach of 24 wells (final 2% Hct at 1 ml) in addition to 30 μl completeRPMI containing recombinant cupredoxin or cytochrome proteins at 666 μMfor a final concentration of 200 μM. Schizont-stage parasites wereprepared by centrifuging a late-stage culture through a Percoll cushionat 3200 rpm for 10 minutes. For infection, 4×10⁶ parasites/well in 500μl volume were added at t=0 hr. The plate was incubated for 30 hours andscored by thin blood smear and Giemsa stain at that time.

The control showed 9.5% parasitemia (standard error 1.3%), wt azurin6.9% (s.e. 1.4%), M44KM64E azurin 9.1% (s.e. 1.0%), rusticyanin 7.2%(s.e. 0.7%), cytochrome c₅₅₁ 7.5% (s.e. 1.5%), human cytochrome c 8.4%(se. 0.40%), plastocyanin 8.1% (s.c. 1.3%) and cytochrome f 6.6% (s.e.1.0%), suggesting that cupredoxins such as wt azurin and rusticyanin andcytochromes such as cytochrome f or cytochrome c₅₅, demonstrated 20 to30% inhibition of parasitemia.

When the cupredoxins were tested for their effects at various stages ofthe parasite life cycle (0-24 hours, ring formation; 24-36 hours,trophozoite; 36-48 hours, schizont), the control showed 0.1% averagering formation and 9.4% trophozoite formation while wt azurin showed noring formation but 6.9% trophozoite formation; cytochrome f showed 0.2%ring formation but had significantly low (6.3%) trophozoite formation.Remarkably, rusticyanin exhibited very high (2.0%) ring formation andsignificantly reduced (5.2%) trophozoite formation. The others had nosignificant effect. The parasites in rusticyanin-treated samples lookedsick and dying as compared to the rest of the samples, showing asignificant inhibitory and toxic effect of rusticyanin on parasitedevelopment.

Example 2 Inhibition In Vitro of P. falciparum Intracellular Replicationby Rusticyanin

To determine if the bacterial redox proteins can inhibit intracellularreplication of the malarial parasites, red blood cells were loaded to anintracellular recombinant protein concentration of 200 μg/ml using ahypotonic ghost preparation. Cells where then washed, resuspended andinfected with schizont-stage parasites (P. falciparum) as described inExample 1. The red blood cell ghosts were incubated for 19 hours and 40hours and giemsa smears were made.

Compared to the infections of normal red blood cells in Example 1, onlyrusticyanin decreased total parasitemia in loaded cell ghost cultures.At 19 hours, there was no significant difference in invasion and ringformation, with empty ghosts at 5.0±0.4% and rusticyanin-loaded ghostsat 4.5±1.0%. However, at 40 hours, rusticyanin-loaded ghosts had a lowerlevel of infection. No major effects were seen at 19 hour with any ofthe bacterial proteins. However, at 40 hours, control untreated ghostsshowed 4.6±0.3% parasitemia while rusticyanin-treated ghosts had2.7±0.8% parasitemia, an almost 50% reduction. See, Table 5. Wt azurin,M44KM64E mutant azurin, plastocyanin, cytochrome c₅₅₁, human cytochromec and cynobacterial cytochrome f proteins showed parasitemia varyingfrom 4.2 to 5.4%.

TABLE 5 Cupredoxin and cytochrome inhibition of P. falciparum infectionof red blood cell ghosts. Mean Parasitemia Treatment at 40 hr Std. ErrorEmpty 4.6% 0.3% Wild Type Azurin 5.4% 1.0% M44KM64E Azurin 4.7% 0.5%Rusticyanin 2.7% 0.8% Cytochrome c₅₅₁ 4.2% 0.4% Human Cytochrome c 4.6%0.8% Plastocyanin 4.3% 0.3% Cytochrome f 4.5% 0.9%

Example 3 Structural Homology Between Azurin and Fab Fragment of G17.12Monoclonal Antibody Complexed with Pf MSP1-19

Previous studies have shown that cupredoxins show structural similarityto the variable domains of the immunoglobulin superfamily members.(Gough & Chothia, Structure 12:917-925 (2004); Stevens et al., J. Mol.Recognit. 18: 150-157 (2005)) The DALI algorithm (Holm & Park,Bioinformatics 16:566-567 (2000)) was used to search the 3D databasesfor structural homologs of azurin (IJZG) from P. aeruginosa. Azurinexhibits structural similarity to the Fab fragment of G17.12 monoclonalantibody in complexation with PfMSP1-19 fragment of the MSP1 merozoitesurface protein of P. falciparum, (Pizarro et al., J. Mol. Biol.328:1091-1103 (2003).) (Table 6) Azurin also exhibits a structuralsimilarity to ICAM-1 (Table 6), which is involved in cerebral malariaand implicated as a receptor on the endothelial cells in themicrovasculture of the brain and other tissues for sequestering P.falciparum-infected erythrocytes. (Smith et al., Proc. Natl. Acad. Sci.USA 97:1766-1771 (2000); Franke-Fayard et al., Proc. Natl. Acad. Sci.USA 102:11468-11473 (2005))

This example shows that cupredoxins including azurin demonstratestructural similarities in having two anti-parallel β sheets packed faceto face and linked by a disulfide bridge to the variable domains of theimmunoglobulin superfamily members as well as extracellular domains ofthe intercellular adhesion molecules (ICAM) and their ligands.

TABLE 6 Structural similarity of P. aeruginosa azurin with variouspathogenesis-related proteins Azurin (1jzg) DALI z PDB AnnotationReference score⁽¹⁾ 1VCA Human Vascular Cell Adhesion 17 3.5 B1Molecle-1, VCAM-1 1ZXQ1 The Crystal Structure of ICAM-2 19 3.3 1IAM1Structure of The Two Amino- 20 3.0 Terminal Domains of, ICAM-1 1OB1Crystal Structure of a Fab complex with 21 2.9 A1 Plasmodium falciparumMSP1-19 1T0P B The complex Structure of Binding 22 2.5 Domains of ICAM-3and Alphabeta2 2NCM Neural Cell Adhesion Molecule, 23 2.4 NCAM⁽¹⁾Structural aligment to azurin were made using DALI (16). Structurepairs with DALI z scores <2 are considered dissimilar.

Example 4 Cloning and Expression of the Laz and H.8-Azurin Fusion Genes

The laz gene from Neisseria gonorrhoeae was cloned based on its knownsequence (SEQ ID NO: 22). The P. aeruginosa azurin gene (SEQ ID NO: 1),termed paz, and the sequence of the H.8 epitope of laz from N.gonnerrhoeae (SEQ ID NO: 21), were used to clone in frame the H.8epitope gene in the 5′-end of paz to produce H.8-paz or in the 3′-end ofpaz to generate paz-H.8.

TABLE 7 Cancer cells, bacterial strains and genetic constructsCells/strains/ plasmids Relevant characteristics* Reference P.aeruginosa Prototroph, FP- (sex factor minus) Holloway, et al., PAO1Microbiol. Rev. 43: 73-102 (1979) E. coli JM109 Cloning and azurinexpression strain Yanisch-Perron, et al., Gene 33: 103-119 (1985) E.coli BL21 GST expression strain Novagen (DE3) N. Prototroph used for DNAisolation American Type Culture gonorrhoeae Collection F62 pUC18 Generalcloning vector, Ap^(r) Yanisch-Perron, et al., id. pUC19 General cloningvector, Ap^(r) Yanisch-Perron, et al., id. pUC18-laz A 1 kb PCR fragmentfrom genomic Herein DNA of N. gonorrhoeae F62 cloned into pUC18pUC19-paz A 0.55 kb PCR fragment from P. Yamada, et al., Proc. Natl.aeruginosa PAO1 cloned into HindIII Acad. Sci. USA 99: 14098- and PstIdigested pUC19, Ap^(r) 14103 (2002); Yamada, et al, Proc. Natl. Acad.Sci. USA 101: 4770-4775 (2004) pUC18-H.8- Fusion plasmid encoding H.8from N. Herein paz gonorrhoeae and azurin from P. aeruginosa PA01,Ap^(r) pGEX-5X-3 GST gene fusion vectors, Ap^(r) Amersham pET29a E. coliexpression vector, Km^(r) Novagen pET29a-gst pET29a derivativecontaining the gst Herein gene, Km^(r) pGEX-5X-3- pGEX-5X-3 derivativecontaining H.8- Herein H.8 encoding region, Ap^(r) pET29a-gst- pET29aderivative containing gst-H.8 Herein H.8 gene, Km^(r) *Ap, ampicillin;Km, kanamycin; GST, Glutathione S-transferase.

Cloning and Expression of the paz and laz Genes.

The cloning and hyperexpression of the azurin gene has been described.(Yamada, et al., Proc. Natl. Acad. Sci. USA 99:14098-14103 (2002); Punj,et al., Oncogene 23:2367-2378 (2004)) The Laz-encoding gene (m/z) ofNeisseria gonorrhoeae was amplified by PCR with genomic DNA of N.gonorrhoeae strain F62 as template DNA. The forward and reverse primersused were

(SEQ ID NO: 23) 5′-CCGGAATTCCGGCAGGGATGTTGTAAATATCCG-3′ and(SEQ ID NO: 24) 5′-CGGGTACCGCCGTGGCAGGCATACAGCATTTCAATCGG-3′where the additionally introduced restriction sites of EcoRI and KpnIsites are underlined respectively. The amplified DNA fragment of 1.0 kb,digested with EcoRI and KpnI, was inserted into the corresponding sitesof pUC18 vector (Yanisch-Perron, et al., Gene 33:103-119 (1985)) so thatthe laz gene was placed downstream of the lac promoter to yield anexpression plasmid pUC18-laz (Table 7).

The plasmids expressing fusion H.8 of N. gonorrhoeae Laz and azurin ofP. aeruginosa (Paz) were constructed by PCR with pUC19-paz and pUC18-lazas templates. For H.8-Paz fusion, a 3.1 kb fragment was amplified withpUC18-laz as a template and primers,5′-(phosphorylated)GGCAGCAGGGGCTTCGGCAGCATCTGC-3′ (SEQ ID NO: 25) and5′-CTGCAGGTCGACTCTAGAGGATCCCG-3′ (SEQ ID NO: 26) where a SalI site isunderlined. A PCR amplified a 0.4 kb fragment was obtained frompUC19-paz as a template and primers,5-(phosphorylated)GCCGAGTGCTCGGTGGACATCCAGG-3′ (SEQ ID NO: 27) and5′-TACTCGAGTCACTTCAGGGTCAGGGTG-3′ (SEQ ID NO: 28) where a XhoI site isunderlined. A SalI digested PCR fragment from pUC18-laz and AhoIdigested PCR fragment from pUC19-paz were cloned to yield an expressionplasmid pUC18-H.8-paz (Table 7).

E. coli JM109 was used as a host strain for expression of azurin and itsderivative genes. Recombinant E. coli strains were cultivated in 2×YTmedium containing 100 μg/ml ampicillin, 0.1 mM IPTG and 0.5 mM CuSO₄ for16 h at 37° C. to produce the azurin proteins.

When E. coli strains harboring these plasmids were grown in presence ofIPTG, cells lysed and the proteins purified as described for azurin(Yamada, et al., Proc. Natl. Acad. Sci. USA 99:14098-14103 (2002); Punj,et al., Oncogene 23:2367-2378 (2004); Yamada, et al., Cell. Microbiol.7:1418-1431 (2005)), the various azurin derivatives migrated on SDS-PAGEas single components, although the H.8 containing proteins (about 17kDa) showed anomalous migrations, as noted before (Cannon, Clin.Microbiol. Rev. 2:S1-S4 (1989); Fisette, et al., J. Biol. Chem.278:46252-46260 (2003)).

Plasmid Construction for Fusion GST Proteins.

Plasmids expressing fusion glutathione S-transferase (GST)-truncatedwt-azurin (azu) derivatives were constructed by a polymerase chainreaction using proofreading DNA polymerase. For pGST-azu 36-128, anamplified PCR fragment was introduced into the BamHI and EcoRI sites ofthe commercial GST expression vector pGEX-5X (Amersham Biosciences,Piscataway, N.J.). The fragment was amplified with pUC19-azu as atemplate and primers, 5′-CGGGATCC CCG GCA ACC TGC CGA AGA ACG TCA TGGGC-3′ (SEQ ID NO: 29) and 5′-CGGAATTC GCA TCA CTT CAG GGT CAG GG-3′ (SEQID NO: 30), where the additionally introduced BamHI and EcoRI sites areunderlined respectively. Carboxyl-terminus truncation of azu gene wascumulatively performed by introducing a stop codon using QuickChangesite-direct mutagenesis kit (Stratagene, La Jolla, Calif.).

For pGST-azu 36-89, a stop codon were introduced into Gly90. The plasmidcarrying pGST-azu 36-128 was used as template DNA. Three sets ofoligonucleotides for site-direct mutagenesis are shown as follows. ForpGST-azu 36-89: 5′-CCA AGC TGA TCG GCT CGT GAG AGAAGG ACT CGG TGA CC-3′(SEQ ID NO: 31), and 5′-GGT CAC CGA GTC CTT CTC TCA CGA GCC GAT CAG CTTGG-3 (SEQ ID NO: 32).

For pGST-azu 88-113, carboxyl terminus truncation of azu gene wascumulatively performed by introducing stop codon using QuickChange sitedirected mutagenesis kit (Stratagene, La Jolla, Calif.). For pGST-azu88-113, a stop codon was introduced into Phe114. The plasmid carryingpGST-azu 88-128 was used as the template. For pGST-azu 88-128 anamplified PCR fragment was introduced into the BamHI and EcoR1 sites ofthe commercial GST expression vector pGEX-5X (Amersham Biosciences). Thefragment was amplified with pUC19-azu as the template and primers,5′-CGGGGATCC CCG GCT CGG GCG AGA AGG AC-3′ (SEQ ID NO: 33) and5′-CGGGAATTC TCC ACT TCA GGG TCA GGG TG-3′ (SEQ ID NO: 34) where theadditionally introduced BamH1 and EcoR1 sites are underlinedrespectively.

One set of oligonucleotides for site directed mutagenesis are shown asfollows for the preparation of pGST-azu 88-113: 5′-GTT CTT CTG CAC CTAGCC GGG CCA CTC CG-3′ (SEQ ID NO: 35) and 5′-CGG AGT GGC CCG GCT AGG TGCAGA AGA AC-3′ (SEQ ID NO: 36). pGST-azu 88-113 was used to transform E.coli XL-1 Blue strains. Plasmid extraction was performed using acommercial kit (Qiagen, Venlo, The Netherlands) and PCR sequencing wereperformed to assess plasmid insertion and transfection.

E. coli BL21 (DE3) was used as a host strain for expression of the gstand its fusions derivatives. E. coli strain XL1-Blue transformed withpGST-azu plasmids was grown in LB media with ampicillin for three hoursat 37° C. upon which IPTG induction (0.4 mM) was performed an subsequentincubation for 24 h at 37° C. to maximize the expression levels. Cellswere isolated by centrifugation, resuspended in 25 mL of 1×PBS buffer.Subsequent cell lysis involved two sequential treatments of the cellsuspension via sonication (20 min on ice) and heat-cold shock inacetone-dry ice bath (using the appropriate protease inhibitors).Supernatants of the cell lysis mixture were isolated and passed througha freshly packed and PBS equilibrated 1 mL glutathione-sepharose 4B(Amersham Biosciences) column. After column washing and subsequentelution of GST-azu product using 10 mM glutathione in 20 mM Tris-HCl pH8. GST-Azu 88-113 purity was tested via electrophoresis using a 10%SDS-PAGE Tris-Gly gel stained with Coomassie Brilliant Blue R reagent.Protein concentration was determined using the Bradford Method.

Example 5 Azurin Binds to the C-Terminal Fragments MSP1-19 and MSP1-42of the P. falciparum Merozoite Surface Protein MSP1

Given the structural similarity (Table 6) between azurin and the fabfragment of the monoclonal antibody G17.12 in complexation with PfMSP1-19 (Pizarro et al., id), the ability of azurin to form a complexwith Pf MSP1-42 or PfMSP1-19 was determined. Two derivatives of azurin,Laz, an azurin-like protein from gonnococci and meningococci such asNeisseria meningitidis with an additional 39 amino acid epitope calledan H.8 epitope (Gotschlich & Seiff, FEMS Microbiol. Lett. 43:253-255(1987); Kawula et al., Mol. Microbiol. 1:179-185 (1987)) and H.8-azurin,where the H.8 epitope of Laz has been fused in the N-terminal part of P.aeruginosa azurin in frame (as described in Example 4) were tested.

In vitro protein-protein interactions were evaluated using a Biacore Xspectrometer from Biacore AB International. All experiments wereconducted at 25° C. in HBS-EP running buffer (0.01 M HEPES, pH 7.4, 0.15M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20) using Au-CM5 sensor chips(Biacore). Protein immobilizations on CM5 chips were conducted accordingto the amine coupling procedure. Proteins were immobilized after NHS/EDCpreactivation of the CM5 surface: 50 μl injections of azurin (510 μM).Subsequent treatment of CM5 surface with ethanolamine (1M, pH 8.8)removed uncrosslinked proteins. Binding studies were performed byinjecting protein eluents (50 μl) over the protein-CM5 surface at flowrates of 30 μl/min with a 120 sec time delay at the end of theinjections. Protein eluents included GST-azurin fusion proteins (GST,GST-Azu 36-128, GST-Azu 36-89, and GST-Azu 88-113, as described inExample 4). Sensor chip surfaces were regenerated between proteininjections using 100 mM NaOH (10 μl injection pulse). All bindingstudies were run in parallel against a negative flow channel with bareAu-CM5 sensor surface to correct for nonspecific binding to the chips.To generate binding constant data, titration experiments were designedvia injection of increasing concentrations of protein eluents (0.05-2000nM). The SPR data were fit to a Langmuir (1:1) equilibrium binding model[Req=Rmax/(1+Kd/C] as specified in the Biacore software from whichbinding constants (Kd) were extrapolated.

Specific interactions of the Pf MSP1-19 and Pf MSP1-42 proteins withazurin, H.8-azurin and Laz were determined by surface plasmon resonance(SPR) analysis and the data are presented in FIG. 1. SPR sensorgrams forbinding of immobilized Pf MSP1-19 and Pf MSP1-42 with azurin and itsderivatives indicated selective recognition among these proteins. Whilenanomolar concentrations of azurin allowed significant binding with theimmobilized MSP1-19 (FIG. 1A) or MSP1-42 (FIG. 1B), both H.8-azurin andLaz demonstrated a higher affinity of binding with the merozoite surfaceprotein MSP1 cleavage products, with characteristic Kd values of 32.2 mMbetween azurin and MSP1-19 and 54.3 nM between azurin and MSP1-42. TheKd values between H.8-azurin and MSP1-19 and MSP1-42 were 11.8 nM and14.3 nM while such values between Laz and MSP1-19 and MSP1-42 rangedfrom 26.2 nM and 45.6 nM respectively.

To examine if the H.8 epitope might facilitate binding of the H.8-azurinor Laz to the PfMSP1-19 or PfMSP1-42 moieties, the ability ofglutathione S-transferase (GST) and a fusion derivative H.8-GST wherethe H.8 epitope was fused in the N-terminal of GST (see Example 4), tobind MSP1-19 or MSP1-42 was tested. Neither the GST nor the H.8-GSTbound PfMSP1-19 (FIG. 1A) or MSP142 (FIG. 1B), although H.8-GST showed aweak binding with MSP1-42.

Glutathione S transferase (GST) and some of the fusion proteins whereparts of azurin were fused to GST (Yamada et al., Cell. Microbiol.7:1418-1431 (2005), and Example 4) were tested for their ability to bindto MSP1-19. GST alone, or GST-Azu 88-113, where the azurin amino acidsequence 88 to 113 out of 128 amino acids of azurin was fused to GST inframe, did not show any binding (FIG. 1C) while GST-Azu 36-89 with aminoacid sequence 36 to 89 and GST-Azu 36-128 with amino acid sequence 36 to128 showed significant binding with MSP1-19 with Kd values of 20.9 nMand 24.5 nM respectively.

Example 6 Inhibition of Plasmodium falciparum Parasitemia by Azurin,H.8-Azurin and Laz

The extent of parasitemia was determined using schizont stage parasitesand normal red blood cells (RBC). Normal red blood cells (RBCs) werewashed twice in serum-free medium and resuspended to 10% hematocrit incomplete RPMI. 200 μl of 10% hematocrit RBCs were added to each of 24wells in addition to 300 μl complete RPM1 without or with azurin,H.8-azurin or Laz at various concentrations. Schizont stage P.falciparum parasites were prepared by centrifuging a late-stage culturethrough a Percoll cushion at 3200 rpm for 10 min. For infection, 4×10⁶parasites per well in 500 μl volume were added at time zero. The platewas incubated overnight (about 16 h) and then scored by thin blood smearand Giemsa stain at that time.

Azurin, H.8-azurin or Laz all demonstrated significant inhibition ofparasitemia in a dose-dependent manner (FIG. 2), although at relativelyhigh concentrations (about 50 μM). Such high concentrations presumablyreflect the multiple ways the malarial parasites invade the erythrocytes(Cowman et al., FEBS Lett. 476:84-88 (2000); Baum et al., J. Biol. Chem.281:5197-5208 (2006)) and a high concentration of azurin or Laz isnecessary to interfere in the entry process. As indicated by theirenhanced binding affinities to MSP1-19, both H.8-azurin and NeisserialLaz protein showed a higher level of inhibition of P. falciparumparasitemia as compared to azurin (FIG. 2).

When azurin was labeled with the red fluorescent dye Alexa fluor 568 andused during the invasion assay, very little red fluorescence wasdetectable inside the RBC, suggesting that azurin seems not to enter theRBC as part of bound MSP1-19, or more likely, that the RBCs that showedthe presence of the schizonts were the ones where azurin failed to bindwith the MSP1-19. These data fully agree with our previous observation(Yamada et al., Cell. Microbiol. 7:1418-1431 (2005)) that azurin doesnot enter normal cells such as macrophages, mast cells, etc, and theeffect of azurin, H.8-azurin or Laz is at the entry level rather thanthe intracellular replication of the parasite. Taken together, the datain FIG. 2 demonstrate the potential antimalarial action of azurin,H.8-azurin and Laz through interference in the invasion of the RBC bythe parasites.

Example 7 Azurin Binds ICAMs

An interesting structural similarity between azurin and ICAMs Table 6)that are known to be involved as receptors for P. falciparum-infectederythrocytes (Wassmer et al., PloS Med. 2:885-890 (2005); Dormeyer etal., Antimicrob. Agents Chemotherap. 50:724-730 (2006)) prompted testanalysis of protein-protein interactions as measured by SPR betweenazurin and ICAMs such as ICAM-1, ICAM-2, ICAM-3 and NCAM. Withimmobilized azurin on the CM5 chip, ICAM-3 (FIG. 3, Kd=19.5±5.4 nM) andNCAM (FIG. 3, inset), but interestingly not ICAM-1 and ICAM-2, showedstrong binding. While not limiting the manner in which the inventionoperates, part of effect of azurin on inhibition of P. falciparumparasitemia might also be mediated through its interaction with ICAM-3or NCAM.

Example 8 Treatment of Patients Likely Exposed to or Exposed to Malaria

Clinical use for the prevention malaria, a pharmaceutical comprising oneor more cupredoxin and/or a cytochrome is administered to a patient.

Fifteen healthy male volunteers, aged 22-50, who have a no history ofpreexisting antibodies to blood-stage P. falciparum parasites, asdetermined by immunofluorescent assay, but reside in an area wheremalaria is endemic, will be injected with a pharmaceutical preparationof purified cupredoxin and purified cytochrome. Two such men will serveas non-treated controls.

The sterile pharmaceutical preparation is in the form of 0.5 mlsingle-dose ampules of sterile Pseudomonas aerugninosa azurin in apharmaceutical preparation designed for intravenous administration, aswill be well known to those in the art. The pharmaceutical preparationis stored at 4° C. and protected from light before administration. Inone clinical trial, azurin is prepared at five different concentrations:10 μg, 30 μg, 100 μg, 300 μg and 800 μg azurin per 0.5 ml dose.

The pharmaceutical preparation is given intravenously to thirteenvolunteers for each 10 doses. Volunteers receive primary treatment atday 0 and subsequent doses identical doses at every other day for threeweeks. Volunteers are observed for immediate toxic effects for twentyminutes after injection. Twenty-four and forty-eight hours later, theyare examined for evidence of fever, local tenderness, erythema, warmth,induration and lymphadenopathy, and are asked about complaints ofheadache, fever, chills, malaise, local pain, nausea and joint pain.Before each dose, blood and urine samples are taken for full laboratoryexamination. Complete blood count and serum chemistry profiles arerechecked two days after each dose. The presence of the malaria parasiteare determined by light microscopic examination (ME) of the stainedblood smears, or the ICT Malaria P.f./P.v. test kits (Binax, Inc.,Portland, Me.). The results demonstrate the effectiveness of thetherapy.

Example 9 Control of Malaria Infection of Insects

A transmissible genetic element that passes from one mosquito to anotherwill be operably connected to the cupredoxin coding sequence operablyconnected to a constitutive promoter. The P. aeruginosa azurin willtherefore be produced inside the Anopheles gambiae infected with P.falciparum and will interfere with its replication/survival in themosquito. This mosquito will then be introduced to an endemic area sothat the azurin-harboring genetic element will spread to other P.falciparum-infected A. gambiae mosquitoes, inhibiting P. falciparumgrowth or survival.

Example 10 Treatment of Patients Infected by Malaria

Clinical use of a malaria therapy, comprising one or more cupredoxinand/or a cytochrome, for treatment of malaria infection in humans.

Fifteen healthy male volunteers, aged 22-50, who exhibit a history ofpreexisting antibodies to blood-stage P. falciparum parasites, asdetermined by immunofluorescent assay are injected with a pharmaceuticalpreparation of purified P. aeruginosa azurin. Two such men serve astreated controls.

The sterile pharmaceutical preparation is in the form of 0.5 mlsingle-dose ampules of sterile P. aeruginosa azurin in a pharmaceuticalpreparation designed for intraveneous administration, as will be wellknown to those in the art. The pharmaceutical preparation is stored at4° C. and protected from light before administration. In one clinicaltrial, P. aeruginosa azurin is prepared at five differentconcentrations: 10 μg, 30 μg, 100 μg, 300 μg and 800 μgazurin/cytochrome c₅₅₁ (1:1 on molecule basis) per 0.5 ml dose.

The pharmaceutical preparation is given intravenously to thirteenvolunteers for each 10 doses. Volunteers receive primary treatment atday 0 and subsequent doses identical doses at every other day for threeweeks. Volunteers are observed for immediate toxic effects for twentyminutes after injection. Twenty-four and forty-eight hours later, theyare examined for evidence of fever, local tenderness, erythema, warmth,induration and lymphadenopathy, and are asked about complaints ofheadache, fever, chills, malaise, local pain, nausea and joint pain.Before each dose, blood and urine samples are taken for full laboratoryexamination. Complete blood count and serum chemistry profiles arerechecked two days after each dose. The presence of the malaria parasiteare determined by light microscopic examination (ME) of the stainedblood smears, or the ICT Malaria P.f./P.v. test kits (Binax, Inc.,Portland, Me.). The results demonstrate the effectiveness of thetherapy.

What is claimed is:
 1. A composition, comprising cytochrome c consistingof SEQ ID NO: 2 or SEQ ID NO: 19 that inhibits parasitemia inmalaria-infected red blood cells, and at least one additional drug in apharmaceutical composition, wherein the at least one additional drug isselected from the group consisting of an anti-malarial drug and ananti-HIV drug.
 2. The composition of claim 1, wherein the pharmaceuticalcomposition is formulated for intravenous administration.